Method for producing preparation containing bioactive substance

ABSTRACT

A method for producing a preparation containing a bioactive substance, characterized in that it comprises forming a solid material containing the bioactive substance and a polymer, and contacting the solid material with a high pressure gas. The method allows the production of a preparation which is suppressed in excessive initial release of the bioactive substance immediately after the administration thereof, is capable of releasing a predetermined amount of the bioactive substance over a long period of time, and is extremely reduced in the deterioration of the bioactive substance and in the amount of a residual organic solvent.

TECHNICAL FIELD

[0001] The present invention relates to a method for producing apreparation containing a bioactive substance. More specifically, thepresent invention relates to a method for producing a preparationcomprising a bioactive substance, which is unstable to heat or solvents,and a polymer.

BACKGROUND ART

[0002] Peptidic or non-peptidic bioactive substances are known toexhibit various pharmacological activities in a living body, and theapplication thereof as medicaments have been attempted. However, it isrequired to administer these bioactive substances frequently since theirhalf-life in a living body is generally short. Then, physical burden ofpatients due to administration by injection cannot be ignored. Forexample, growth hormone, a representative hormone which is originallyproduced and secreted in the anterior portion of the pituitary gland, isa bioactive peptide having widely diverse physiological activities suchas, in addition to promotion of growth in the body, metabolism ofsaccharides and lipids, anabolism of proteins, cell proliferation anddifferentiation, and the like. At present, growth hormone is produced ona large scale by Escherichia coli using genetic recombinationtechnology, and put to medicinal use clinically and worldwide. However,it is required to administer growth hormone frequently in order tomaintain an effective blood level because of its short biologicalhalf-life. Especially, in the case of pituitary dwarfism, a dailysubcutaneous administration to infants or young patients over a longperiod of time ranging from a few months to 10 years or more is actuallytaken place.

[0003] In order to deal with problems inherent in such bioactivesubstances, various drug delivery systems have been studied. Forexample, a sustained-release agent that provides sustained-release of abioactive peptide over a long period of time has been studied. JP8-217691 A (WO96/07399) discloses a method for producing asustained-release preparation comprising a water-insoluble or poorlywater soluble polyvalent metal salt of a water-soluble peptidicbioactive substance, which is formed by an aqueous solution of zincchloride, etc., and a biodegradable polymer.

[0004] Further, for a sustained-release preparation using abiodegradable polymer, it is desired to maintain the activity of abioactive substance with suppressing the initial release of a bioactivesubstance, in particular, release of the excess amount within one day,and to control the release of the bioactive substance arbitrarily over along period of time. Regarding this problem, JP 11-322631 A discloses amethod for producing a sustained-release preparation comprising adding awater-miscible organic solvent and/or a volatile salt to an aqueoussolution of a bioactive peptide, followed by lyophilizing to obtain abioactive peptide powder, dispersing the powder in a solution of abiodegradable polymer in an organic solvent, and removing the organicsolvent. Furthermore, JP 9-132524 A discloses a method for producingsustained-release microcapsules comprising a bioactive substance and abiodegradable polymer which comprises, after forming microcapsules,heat-drying the microcapsules at a temperature of not less than theglass transition temperature of the biodegradable polymer for about 24to 120 hours. These are methods for producing a sustained-releasepreparation containing very little residual organic solvent and havingvery superior clinical properties as medicaments.

OBJECTS OF THE INVENTION

[0005] However, according to the solvent-removing procedures in theabove-mentioned production methods, since it takes long period of timefor removing the solvent, there is still room for improvement in view ofthe production costs for the industrial application.

[0006] On the other hand, as a procedure for removing a solvent thatremains in a component (e.g., polymer) used for formulating apreparation of a medicament, heat drying method, vacuum drying methodand flash drying with dried gas have been known. However, in theseprocedures, when a substance has strong affinity for a solvent and isunstable to heat, the removal of the solvent tends to be insufficientor, in some cases, the substance is decomposed. Furthermore, in theseprocedures, when the boiling point of a solvent to be removed is high,the properties of a preparation obtained may be deteriorated.

SUMMARY OF THE INVENTION

[0007] The present inventors have studied intensively to solve theabove-mentioned problems and unexpectedly found that, in a method forproducing a sustained-release preparation comprising a bioactivesubstance and a biodegradable polymer, a sustained-release preparationhaving superior clinical properties as a medicament, in which excessinitial release of the bioactive substance immediately afteradministration is markedly suppressed, a constant amount of thebioactive substance is being released from immediately afteradministration over a long period of time and very little residualorganic solvent is contained therein, can be obtained by, after forminga solid material, contacting the solid material with high-pressure gasfor about 10 minutes to about 12 hours. The present invention has beencompleted based on these findings.

[0008] That is, the present invention provides:

[0009] (1) A method for producing a preparation containing a bioactivesubstance, which comprises forming a solid material containing thebioactive substance and a polymer, and contacting the solid materialwith high-pressure gas;

[0010] (2) The method according to the above (1), wherein the bioactivesubstance is that being unstable to heat or solvents;

[0011] (3) The method according to the above (1), wherein the bioactivesubstance is a bioactive peptide having a molecular weight of about2,000 to about 500,000;

[0012] (4) The method according to the above (1), wherein the bioactivesubstance is a bioactive peptide having a molecular weight of about5,000 to about 500,000;

[0013] (5) The method according to the above (4), wherein the bioactivesubstance is human growth hormone;

[0014] (6) The method according to the above (1), wherein the bioactivesubstance is a non-peptidic compound;

[0015] (7) The method according to the above (6), wherein thenon-peptidic compound is a compound having an oxygen atom in themolecule;

[0016] (8) The method according to the above (6), wherein thenon-peptidic compound is a compound having an ether bond or a carbonylgroup;

[0017] (9) The method according to the above (6), wherein thenon-peptide compound is a compound represented by the formula (I):

[0018] wherein R¹ represents a group capable of forming an anion or agroup which may be converted into said group, X represents that thephenylene group and the phenyl group are linked directly or via a spacerof an atomic chain having two or less atom(s), n represents an integerof 1 or 2, ring A represents a benzene ring which may be furthersubstituted, R² represents a group capable of forming an anion or agroup which may be converted into said group, R³ represents ahydrocarbon residue which may link via a heteroatom and may besubstituted, or a salt thereof;

[0019] (10) The method according to the above (6), wherein thenon-peptidic compound is losartan, eprosartan, candesartan cilexetil,candesartan, valsartan, telmisartan, irbesartan, tasosartan orolmesartan;

[0020] (11) The method according to the above (6), wherein thenon-peptide compound is candesartan;

[0021] (12) The method according to the above (1), wherein the polymeris biodegradable;

[0022] (13) The method according to the above (12), wherein thebiodegradable polymer is a homopolymer or a copolymer ofα-hydroxycarboxylic acids, or a mixture thereof;

[0023] (14) The method according to the above (13), wherein thebiodegradable polymer is a homopolymer or a copolymer of lacticacid/glycolic acid having a composition ratio of lactic acid/glycolicacid of about 100/0 to about 40/60 mol %;

[0024] (15) The method according to the above (13), wherein thebiodegradable polymer is a homopolymer of lactic acid;

[0025] (16) The method according to the above (12), wherein theweight-average molecular weight of the biodegradable polymer is about3,000 to about 50,000;

[0026] (17) The method according to the above (1), wherein the solidmaterial is contacted with high-pressure gas at a temperature range ofabout +20° C. to about −60° C. based on the glass transition temperatureof the polymer;

[0027] (18) The method according to the above (17), wherein the solidmaterial is contacted with high-pressure gas at a temperature range ofabout +0° C. to about −40° C. based on the glass transition temperatureof the polymer;

[0028] (19) The method according to the above (1), wherein the periodfor contacting the solid material with high-pressure gas is about 5minutes to about 48 hours;

[0029] (20) The method according to the above (19), wherein the periodfor contacting the solid material with high-pressure gas is about 10minutes to about 12 hours;

[0030] (21) The method according to the above (1), wherein thehigh-pressure gas is inert to the bioactive substance and polymer;

[0031] (22) The method according to the above (21), wherein thehigh-pressure gas is carbon dioxide;

[0032] (23) The method according to the above (1), wherein the pressureof the high-pressure gas is about 1 MPa to about 7 MPa;

[0033] (24) The method according to the above (23), wherein the pressureof the high-pressure gas is about 1 MPa to about 4 MPa;

[0034] (25) The method according to the above (23), wherein thepreparation is sustained-release microcapsules;

[0035] (26) The method according to the above (25), wherein thesustained-release microcapsules are obtained by drying-in-water method;

[0036] (27) A preparation obtained by the method according to the above(1);

[0037] (28) Sustained-release microcapsules obtained by the methodaccording to the above (25);

[0038] (29) An injectable preparation comprising the sustained-releasemicrocapsules according to the above (28);

[0039] (30) A method for suppressing the initial release of a bioactivesubstance, which comprises forming a solid material containing saidbioactive substance and a polymer, and contacting the solid materialwith high-pressure gas; and

[0040] (31) A method for suppressing the denaturation of a bioactivesubstance, which comprises forming a solid material containing saidbioactive substance and a polymer, and contacting the solid materialwith high-pressure gas.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1 is a schematic drawing of a solvent-removing apparatususing carbon dioxide in a high-pressure gaseous state.

[0042] Each number in the drawing has the following meaning.

[0043] 1: liquefied carbon dioxide bomb, 2: CO₂ delivery pump, in-water3: heat exchanger, 4: extraction vessel, 5: thermostat, 6: detector, 7:automatic pressure regulating valve, 8: recovery vessel.

DETAILED DESCRIPTION OF THE INVENTION

[0044] The bioactive substance used in the present invention includesvarious drugs, which have useful physiological activities for animalsand plants and can be used as an agrochemical or an animal drug, or canbe used clinically. As the bioactive substance used in the presentinvention, a bioactive substance being unstable to heat or solvents ispreferred. The bioactive substance being unstable to heat or solventsused herein means a bioactive substance that is decomposed, metabolized,inactivated or denatured in a production step which involves heating orcontact with an organic solvent, such as emulsification, removing of asolvent or drying. The agrochemical includes, for example, controlagents for pests, control agents for plant diseases, herbicides, plantgrowth regulators, fertilizers and the like, and the animal drugincludes, for example, antibacterial agents, vitamin preparations,hormone preparations, vaccines, additives for fishery products,insecticide and disinfectant preparations, drugs for pets and the like.For an ideal agrochemical or animal drug, which is safe andenvironment-friendly, reduction of residual solvents is important.Examples of various drugs those can be used clinically include, and arenot specifically limited, peptidic compounds having physiologicalactivities, as well as antibiotics, antifungal agents,antihyperlipidemic agents, antitumor agents, antipyretic agents,analgesic agents, antiinflammatory agents, antitussive and expectorantagents, sedatives, muscle relaxants, antiepileptics, antiulcer agents,antidepressants, antiallergic agents, cardiotonics, antiarrhythmicagents, vasodilators, hypotensive diuretics, antidiabetic agents,anticoagulants, hemostatic agents, antiplatelet agents, antituberculousagent, hormones, antinarcotics, bone resorption-suppressing agents,osteogenesis-accelerating agents, and neovascularization suppressingagents. Of these, a peptidic or non-peptidic bioactive substance thatproduces a dimer, a polymer or related substances such as an oxidizedsubstances, deamidated substances, and the like by heat, or a peptidicor non-peptidic bioactive substance that produces a reaction productwith a biodegradable polymer, is preferably used in the presentinvention.

[0045] The bioactive peptide in the present invention includes variouspeptides or proteins, which have physiological activities useful formammals and can be used clinically. As the “bioactive peptide”, thathaving a molecular weight (as monomers) of, for example, about 200 to500,000, preferably molecular weight of about 2,000 to 500,000, isgenerally used. More preferably, a peptide having a molecular weight of5,000 to about 500,000 is used.

[0046] The typical activity of the bioactive peptide includes hormoneactivities. The bioactive peptide may be any of natural substances,synthesized substances and semi-synthesized substances, or may bederivatives or related substances thereof. The functional mechanism ofthe bioactive peptide may be either of agonistic and antagonistic.

[0047] As the bioactive peptide in the present invention, there can beused peptide hormones, cytokines, peptide nerve transmitter substances,hematopoietic factors, various growth factors, enzymes, polypeptideantibiotics, analgetic peptides, vaccines, and the like.

[0048] As the peptide hormones, there can be used insulin, somatostatin,somatostatin derivatives (Sandostatin; see U.S. Pat. Nos. 4,087,390,4,093,574, 4,100,117 and 4,253,998), growth hormones (GH), sodiumdiuretic peptides, gastrin, prolactin, adrenocorticotropic hormone(ACTH), ACTH derivatives (e.g., ebiratide, etc.), melanocyte-stimulatinghormone (MSH), thyroid hormone-releasing hormone (TRH) and salts andderivatives thereof (see JP 50-121273 A and JP 52-116465 A),thyroid-stimulating hormone (TSH), luteinizing hormone (LH),follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG),thymosin, motilin, vasopressin, vasopressin derivatives [desmopressin,see Folia Endocrinologica Japonica, Vol. 54, No. 5, pp. 676-691 (1978)],oxytocin, calcitonin, parathyroid hormone (PTH), glucagon, secretin,pancreozymin, cholecystokinin, angiotensin, human placental lactogen,glucagon-like peptide (GLP-1) and derivatives thereof (see JP 6-80584 A,JP 7-2695 A, EP 658568, JP 8-245696 A, JP 8-269097 A, WO97/15296,WO97/31943, WO98/19698, WO98/43658, JP 10-511365 A, WO99/55310, JP11-513983 A, CA2270320, WO99/64061, JP 11-514972 A, JP 2000-500505 A,WO2000/66138, WO2000/66142, WO2000/78333, JP 2001-11095, Tissue Eng.7(1)35-44(2001), Diabetologia 43(10)1319-1328(2000), WO2000/34331,WO2000/34332, U.S. Pat. No. 6,268,343, U.S. 2001011071 A, U.S.2001006943 A, EP 0733644, WO2000/77039, WO99/43707, WO99/43341,WO99/43706, WO99/43708, WO99/43705, WO99/29336, WO2000/37098, EP0969016, U.S. Pat. No. 5,981,488, U.S. Pat. No. 5,958,909, WO93/25579,WO98/43658, EP 0869135, U.S. Pat. No. 5,614,492, U.S. Pat. No.5,545,618, U.S. Pat. No. 5,120,712, U.S. Pat. No. 5,118,666, WO95/05848,WO91/11457, EP 0708179, WO96/06628, EP0658568, WO87/06941),glucose-dependent insulin secretory peptide (GIP), exendin andderivatives thereof (see WO2000/66629, WO2000/41546, WO99/07404,WO2000/09666, and U.S. Pat. No. 5,424,286), metastin and derivativesthereof (see WO2000/24890), and the like. Preferably, the peptidehormone is insulin and growth hormone, etc.

[0049] Growth hormone (hereinafter referred to as GH) originating fromany animal species can be used, and is preferably human growth hormone(hereinafter referred to as hGH). Further, although natural productsextracted from the pituitary gland can be used in the present invention,genetic recombinant type GH (see JP 6-12996 B and JP 6-48987 B) ispreferred. The recombinant type hGH having the same structure as anatural type which does not have methionine at the N-terminal group ismore preferred. Such GH may be in the form of a metal salt, and onebeing substantially free from a metal is also used. About 20K daltontype of hGH (see JP 7-101877 A and JP 10-265404 A) as well as about 22Kdalton type of hGH can be used. Furthermore, the derivatives or relatedsubstances of hGH (see WO99/03887) can be used.

[0050] As the cytokines, for example, lymphokines, monokines, and thelike can be used. As the lymphokines, there can be used, for example,interferons (alpha type, beta type, gamma type and the like),interleukins (IL-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and the like) andthe like. As the monokines include, for example, interleukin-1 (IL-1),tumor necrosis factor (TNF), and the like can be used. The cytokines arepreferably lymphokines, and the like, more preferably interferons, andthe like, especially preferably interferon-alpha.

[0051] As the peptide neurotransmitters, substance P, serotonin, GABA,and the like can be used.

[0052] As the hematopoietic factors, there can be used, for example,erythropoietin (EPO), colony stimulating factors (G-CSF, GM-CSF, M-CSFand the like), thrombopoietin (TPO), platelet growth stimulating factor,megakaryocyte potentiator, and the like.

[0053] As various growth factors, there can be used, for example, basicand acidic fibroblast growth factors (FGF) and their family (e.g., EGF,TGF-α, TGF-β, PDGF, acidic FGF, basic FGF, FGF-9, and the like), nervegrowth factors (NGF) and their family (e.g., BDNF, NT-3, NT-4, CNTF,GDNF, and the like), insulin-like growth factors (e.g. IGF-1, IGF-2, andthe like), bone morphogenetic protein (BMP) and their family, and thelike.

[0054] As the enzymes include, there can be used, for example,superoxide dismutase (SOD), urokinase, tissue plasminogen activator(TPA), asparaginase, kallikrein, and the like.

[0055] As the polypeptide antibiotics, for example, polymixin B,colistin, gramicidin, bacitracin, and the like can be used.

[0056] As the analgesic peptides, for example, enkephalin, enkephalinderivatives (see U.S. Pat. No. 4,277,394 and EP 031567 A), endorphin,kyotorphin, and the like can be used.

[0057] As the vaccines, there can be used, for example, influenzavaccine, Japanese encephalitis vaccine, antirabies vaccine, hepatitis Bvaccine, hepatitis A vaccine, cholera vaccine, DPT mixed vaccine,pneumococcus vaccine, diphteria vaccine, tetanus vaccine, polio vaccine,prostatic specific antigen vaccine, and the like.

[0058] Furthermore, the bioactive peptides include thymopoietin,dynorphin, bombesin, caerulein, thymostimulin, thymic humoral factor(THF), blood thymic factor (FTS) and derivatives thereof (see U.S. Pat.No. 4,229,438), other thymic factors [Igaku no Ayumi, Vol.125, No. 10,pp. 835-843 (1983)], neurotensin, bradykinin, endothelin-antagonisticpeptides (see EP 436189 A, EP 457195 A and EP 496452 A, JP 3-94692 A andJP 3-130299 A), and the like.

[0059] In the present invention, when the bioactive peptide contains ametal, the metal contained in the bioactive peptide may be removedpreviously, if necessary, and, as a method for removing the metal, aknown method can be used. For example, an insulin in the form ofamorphous and containing a minimum amount of metal can be obtained bydialyzing an aqueous solution of insulin acidified with hydrochloricacid to water or a solution of ammonium acetate, followed bylyophilization.

[0060] As the non-peptidic bioactive substance of the present invention,for example, there may be mentioned one or more components selected fromnourishing and health-promoting agents,antipyretic-analgesic-antiinflammatory agents, antipsychotic drugs,antianxiety drugs, antidepressants, hypnotic-sedatives, spasmolytics,central nervous system affecting drugs, cerebral metabolismameliolators, antiepileptics, sympathomimetic agents, gastrointestinalfunction conditioning agents, antacids, antiulcer agents,antitussive-expectorants, antiemetics, respiratory stimulants,bronchodilators, antiallergic agents, dental buccal drugs,antihistamines, cardiotonics, antiarrhythmic agents, diuretics,hypotensive agents, vasoconstrictors, coronary vasodilators, peripheralvasodilators, antihyperlipidemic agents, cholagogues, antibiotics,chemotherapeutic agents, antidiabetic agents, drugs for osteoporosis,skeletal muscle relaxants, antimotion sickness drugs, hormones, alkaloidnarcotics, sulfa drugs, drugs for treatment of gout, anticoagulants,anti-malignant tumor agents, agents for Alzheimer's disease and thelike.

[0061] Examples of the nourishing and health-promoting agents includevitamins such as vitamin A, vitamin D, vitamin E (d-α-tocopherol acetateand the like), vitamin B₁ (dibenzoylthiamine, fursultiaminehydrochloride and the like), vitamin B₂ (riboflavin butyrate and thelike), vitamin B₆ (pyridoxine hydrochloride and the like), vitamin C(ascorbic acid, sodium L-ascorbate and the like), vitamin B₁₂(hydroxocobalamin acetate and the like) and the like; minerals such ascalcium, magnesium and iron; amino acids; oligosaccharides; galenical;and the like. Examples of the antipyretic-analgesic-antiinflammatoryagents include aspirin, acetaminophen, ethenzamide, ibuprofen,diphenhydramine hydrochloride, dl-chlorpheniramine maleate,dihydrocodeine phosphate, noscapine, methylephedrine hydrochloride,phenylpropanolamine hydrochloride, caffeine, anhydrous caffeine,tolfenamic acid, mefenamic acid, diclofenac sodium, flufenamic acid,salicylamide, aminopyrine, ketoprofen, indomethacin, bucolome,pentazocine and the like. Examples of the antipsychotic drugs includechlorpromazine, reserpine and the like. Examples of the antianxietydrugs include alprazolam, chlordiazepoxide, diazepam and the like.Examples of the antidepressants include imipramine, maprotiline,amphetamine and the like.

[0062] Examples of the hypnotic-sedatives include estazolam, nitrazepam,diazepam, perlapine, phenobarbital sodium and the like. Examples of thespasmolytics include scopolamine hydrobromide, diphenhydraminehydrochloride, papaverine hydrochloride and the like. Examples of thecentral nervous system affecting drugs include citicoline, rotirenineand the like. Examples of the cerebral metabolism ameliolators includevinpocetine, meclofenoxate hydrochloride and the like. Examples of theantiepileptics include phenytoin, carbamazepine and the like. Examplesof the sympathomimetic agents include isoproterenol hydrochloride andthe like. Examples of the gastrointestinal function conditioning agentsinclude stomachic-digestives such as gentian, swertia herb, nux vomica,phellodendron bark, bitter orange peel, Condurango, cinnamon oil and thelike; intestinal function controlling drugs such as perperinehydrochloride, resistant lactic acid bacterium, Lactobacillus bifidusand the like. Examples of the antacids include magnesium carbonate,sodium hydrogen carbonate, magnesium aluminometasilicate, synthetichydrotalcite, precipitated calcium carbonate, magnesium oxide and thelike. Examples of the antiulcer agents include lansoprazole, omeprazole,rabeprazole, pantoprazole, famotidine, cimetidine, ranitidinehydrochloride and the like.

[0063] Examples of the antitussive-expectorants include chloperastinehydrochloride, dextromethorphan hydrobromide, theophylline, potassiumguaiacolsulfonate, guaifenesin, codeine phosphate and the like. Examplesof the antiemetics include diphenidol hydrochloride, metoclopramide andthe like. Examples of the respiratory stimulants include levallorphantatrate and the like. Examples of the bronchodilators includetheophylline, salbutamol sulfate and the like. Examples of theantiallergic agents include amlexanox, seratrodast and the like.Examples of the dental buccal drugs include oxytetracycline,triamcinolone acetonide, chlorhexidine hydrochloride, lidocaine and thelike. Examples of the antihistamines include diphenhydraminehydrochloride, promethazine, isothipendyl hydrochloride,dl-chlorpheniramine maleate and the like. Examples of the cardiotonicsinclude caffeine, digoxin and the like. Examples of the antiarryhythmicagents include procainamide hydrochloride, propranolol hydrochloride,pindolol and the like. Examples of the diuretics include isosorbide,furosemide and the like. Examples of the hypotensive agents includedelapril hydrochloride, captopril, hexamethonium bromide, hydralazinehydrochloride, labetalol hydrochloride, manidipine hydrochloride,losartan, eprosartan, candesartan cilexetil (TCV-116), candesartan(CV-11974), valsartan, telmisartan, irbesartan, tasosartan, ormesartanand the like. As the hypotensive agents, candesartan, candesartancilexetil and the like are preferred, and candesartan and the like arespecifically preferred.

[0064] Examples of the vasoconstrictors include phenylephrinehydrochloride and the like. Examples of the coronary vasodilatorsinclude carbocromen hydrochloride, molsidomine, verapamil hydrochlorideand the like. Examples of the peripheral vasodilators includecinnarizine and the like. Examples of the antihyperlipidemic agentsinclude cerivastatin sodium, simvastatin, pravastatin sodium and thelike. Examples of the cholagogues include dehydrocholic acid,trepibutone and the like. Examples of the antibiotics include cephemantibiotics such as cefalexin, amoxicillin, pivmecillinam hydrochloride,cefotiam dihydrochloride, cefozopran hydrochloride, cefinenoximehydrochloride, cefsluodin sodium, etc.; synthetic antibacterials such asampicillin, cyclacillin, sulbenicillin sodium, nalidixic acid, enoxacin,etc.; monobactam antibiotics such as carumonam sodium; penemantibiotics, carbapenem antibiotics, etc.; and the like. Examples of thechemotherapeutic agents include sulfamethizole hydrochloride,thiazosulfone and the like. Examples of the antidiabetic agents includetolbutamide, voglibose, pioglitazone (hydrochloride), troglitazone,5-[[4-[2-(methyl-2-pyridinylamino)ethoxy]phenyl]methyl]-2,4-thiazolidinedione(BRL-49653), acarbose, miglitol, emiglitate and the like. Examples ofthe drugs for osteoporosis include ipriflavone and the like. Examples ofthe skeletal muscle relaxants include methocarbamol and the like.Examples of the antimotion sickness drugs include meclizinehydrochloride, dimenhydrinate and the like.

[0065] Examples of the hormones include riothyroinine sodium,dexamethasone sodium phosphate, prednisolone, oxendolone, leupororelinacetate and the like. Examples of the alkaloid narcotics include opium,morphine hydrochloride, ipecac, oxycodone hydrochloride, opium alkaloidshydrochlorides, cocaine hydrochloride and the like. Examples of thesulfa drugs include sulfanilamide, sufamethizole and the like. Examplesof the drugs for treatment of gout include allopurinol, colchicine andthe like. Examples of the anticoagulants include dicoumarol and thelike. Examples of the anti-malignant tumor agents include5-fluorouracil, uracil, mitomycin and the like. Examples of the agentsfor Alzheimer's disease include idebenone, vinpocetine and the like.

[0066] As the non-peptidic bioactive substance in the present invention,a compound having an oxygen atom in the molecule, specifically, acompound having an ether bond or a carbonyl group is preferred. Suchcompound includes a compound represented by the formula (I):

[0067] or a salt thereof.

[0068] In the above-mentioned formula (I), examples of the group capableof forming an anion (a group having hydrogen atom that may be liberatedas a proton) as R¹ include (1) a carboxyl group, (2) a tetrazolyl group,(3) a trifluoromethanesulfonic acid amide group (—NHSO₂CF₃), (4) aphosphoric acid group, (5) a sulfonic acid group, (6) an optionallysubstituted 5- to 7-membered (preferably 5- or 6-membered) monocyclicheterocycle residue containing one or two or more of N, S and O, and thelike.

[0069] Examples of the above-mentioned “optionally substituted 5- to7-membered (preferably 5- or 6-membered) monocyclic heterocycle residuecontaining one or two or more of N, S and O” include

[0070] and the like. Furthermore, when the g in the above-mentionedformula represents —NH— and the like, the linkage between theheterocyclic residue represented by R¹ and the phenyl group to which theheterocyclic residue is linked, may link via one of existing pluralnitrogen atoms, in addition to the above-mentioned carbon-carbonbond(s). For example, when R¹ is represented by the formula:

[0071] specifically, examples thereof are

[0072] and the like, respectively. The other examples of the link via anitrogen atom include

[0073] and the like.

[0074] In the above-mentioned formula, g represents —CH₂—, —NH—, —O— or—S(O)_(m)—, >=Z, >=Z′ and >=Z″ each represents a carbonyl group, athiocarbonyl group or an optionally oxidized sulfur atom (e.g., S, S(O),S(O)₂ and the like) (preferably a carbonyl or a thiocarbonyl group, morepreferably a carbonyl group), respectively, and m represents 0, 1 or 2.

[0075] The heterocyclic residue represented by R¹ is preferably groupsobtained by eliminating one hydrogen atom from a ring that has both —NH—or —OH group as a proton donor, and a carbonyl group, a thiocarbonylgroup, a sulfinyl group, or the like as a proton acceptor,simultaneously, such as an oxadiazolone ring, an oxadiathiazolone ring,a thiadiazolone group, or the like. Furthermore, the heterocyclicresidue represented by R¹ may form a condensed ring group by linkingsubstituents on the ring. As the heterocyclic residue represented by R¹,a 5- or 6-membered ring residue is preferred, and a 5-membered residueis more preferred.

[0076] As the heterocyclic residue represented by R¹, a grouprepresented by the formula:

[0077] wherein i represents —O— or —S—, j represents >=O, >=S or>=S(O)_(m), m is as defined above (among these,2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-yl,2,5-dihydro-5-thioxo-1,2,4-oxadiazol-3-yl,2,5-dihydro-5-oxo-1,2,4-thiadiazol-3-yl, especially2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-yl) and the like are preferred.

[0078] Furthermore, the above-mentioned heterocyclic residue (R¹) hastautomers as shown below. For example, when Z=O and g=O in the followingformula:

[0079] three tautomers a′, b′ and c′, such as

[0080] are present, and the heterocyclic residue represented by theformula:

[0081] encompasses all of the above-mentioned a′, b′ and c′.

[0082] The group capable of forming an anion as R¹ may be protected withan optionally substituted lower (C₁₋₄) alkyl group or an acyl group(e.g., a lower (C₂₋₅) alkanoyl, a benzoyl, etc.), and the like, atsubstitutable position(s).

[0083] The optionally substituted lower (C₁₋₄) alkyl group include, forexample, (1) a lower (C₁₋₄) alkyl group optionally substituted with 1 to3 phenyl group(s) optionally having a halogen atom, nitro, lower (C₁₋₄)alkyl, lower (C₁₋₄) alkoxy and the like (e.g., methyl, triphenylmethyl,p-methoxybenzyl, p-nitrobenzyl and the like), (2) a lower (C₁₋₄)alkoxy-lower (C₁₋₄) alkyl group (e.g., methoxymethyl, ethoxymethyl andthe like), (3) the formula —CH(R⁴)-OCOR⁵ [wherein R⁴ represents (a)hydrogen, (b) a straight chain or branched lower alkyl group having 1-6carbon atom(s) (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl and the like), (c) astraight chain or branched lower alkenyl group having 2-6 carbon atomsor (d) a cycloalkyl group having 3-8 carbon atoms (e.g., cyclopentyl,cyclohexyl, cycloheptyl and the like), R⁵ represents (a) a straightchain or branched lower alkyl group having 1-6 carbon atom(s) (e.g.,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,t-butyl, n-pentyl, isopentyl, neopentyl and the like), (b) a straightchain or branched lower alkenyl group having 2-6 carbon atoms, (c) alower alkyl group having 1 to 3 carbon atom(s) substituted with acycloalkyl group having 3-8 carbon atoms (e.g., cyclopentyl, cyclohexyl,cycloheptyl and the like) or an optionally substituted aryl group (e.g.,a phenyl or naphthyl group and the like, each of which may have ahalogen atom, nitro, lower (C₁₋₄) alkyl, lower (C₁₋₄) alkoxy and thelike) (e.g., benzyl, p-chlorobenzyl, phenethyl, cyclopentylmethyl,cyclohexylmethyl and the like), (d) a lower alkenyl group having 2 to 3carbon atoms substituted by a cycloalkyl having 3-8 carbon atoms or anoptionally substituted aryl group (e.g., a phenyl or naphthyl group andthe like, each of which may have a halogen atom, nitro, lower (C₁₋₄)alkyl, lower (C₁₋₄) alkoxy and the like) (e.g., a group having alkenylportion(s) such as vinyl, propenyl, allyl, isopropenyl and the like,such as cinnamyl, and the like), (e) an optionally substituted arylgroup (e.g., a phenyl or a naphthyl group and the like, each of whichmay have a halogen atom, nitro, lower (C₁₋₄) alkyl, lower (C₁₋₄) alkoxyand the like, such as phenyl, p-tolyl, naphthyl and the like), (f) astraight or a branched lower alkoxy group having 1-6 carbon atom(s)(e.g., methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy,sec-butoxy, t-butoxy, n-pentyloxy, isopentyloxy, neopentyloxy and thelike), (g) a straight chain or branched lower alkenyloxy group having 2to 8 carbon atoms (e.g., allyloxy, isobutenyloxy and the like), (h) acycloalkyloxy group having 3-8 carbon atoms (e.g., cyclopentyloxy,cyclohexyloxy, cycloheptyloxy and the like), (i) a lower alkoxy grouphaving 1 to 3 carbon atom(s) substituted with a cycloalkyl having 3-8carbon atoms (e.g., cyclopentyl, cyclohexyl, cycloheptyl and the like)or an optionally substituted aryl group (e.g., a phenyl or naphthylgroup and the like, each of which may have a halogen atom, nitro, lower(C₁₋₄) alkyl, lower (C₁₋₄) alkoxy and the like) (e.g., a group havingalkoxy portion(s) such as methoxy, ethoxy, n-propoxy, isopropoxy and thelike, such as benzyloxy, phenethyloxy, cyclopentylmethoxy,cyclohexylmethoxy and the like), (j) a lower alkenyloxy group having 2to 3 carbon atoms substituted with a cycloalkyl having 3-8 carbon atoms(e.g., cyclopentyl, cyclohexyl, cycloheptyl and the like) or anoptionally substituted aryl group (e.g., a phenyl or naphthyl group andthe like, each of which may have a halogen atom, nitro, lower (C₁₋₄)alkyl, lower (C₁₋₄) alkoxy and the like) (e.g., a group havingalkenyloxy portion(s) such as vinyloxy, propenyloxy, allyloxy,isopropenyloxy and the like, such as cinnamyloxy and the like, and thelike), (k) an optionally substituted aryloxy group (e.g., phenoxy ornaphthoxy group and the like, each of which may have a halogen atom,nitro, lower (C₁₋₄) alkyl, lower (C₁₋₄) alkoxy and the like, such asphenoxy, p-nitrophenoxy, naphthoxy and the like, and the like)], and thelike.

[0084] Furthermore, the group capable of forming an anion as R¹ may havesubstituent(s) such as an optionally substituted lower (C₁₋₄) alkylgroup (which includes the groups similar to the “optionally substitutedlower (C₁₋₄) alkyl group” that is exemplified as protective groups forthe group capable of forming an anion as the above-mentioned R¹), ahalogen atom, nitro, cyano, lower (C₁₋₄) alkoxy, an amino optionallysubstituted with 1 or 2 of lower (C₁₋₄) alkyl(s) and the like, at thesubstitutable position(s), in addition to the protective groups such asthe above-mentioned optionally substituted lower (C₁₋₄) alkyl group oracyl group (e.g., a lower (C₂₋₅) alkanoyl, a benzoyl and the like).

[0085] In the above-mentioned formula, the group which may be convertedinto the group capable of forming an anion as R¹ (a group havinghydrogen atom that may be liberated as a proton) may be a group whichcan be converted into the group capable of forming an anion by areaction under biological, i.e., physiological conditions (for example,an in vivo reaction such as oxidation, reduction, hydrolysis or the likewith an in vivo enzyme, and the like) (so-called prodrug), or may be agroup which can be converted into a group capable of forming an anionrepresented by R¹ by a chemical reaction, such as cyano, anN-hydroxycarbamimidoyl group (—C(=N—OH)—NH₂), or (1) a carboxyl group,(2) a tetrazolyl group, (3) a trifluoromethanesulfonic acid amide group(—NHSO₂CF₃), (4) a phosphoric acid group, (5) a sulfonic acid group, and(6) an optionally substituted 5- to 7-membered (preferably 5-or6-membered) monocyclic heterocycle residue containing one or two or moreof N, S and O, each of which has been protected with an optionallysubstituted lower (C₁₋₄) alkyl group or an acyl group (so-calledsynthetic intermediate).

[0086] Preferred examples of R¹ include a carboxyl, a tetrazolyl, or a2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-yl (preferably tetrazolyl)optionally protected with an optionally substituted lower (C₁₋₄) alkyl(e.g., methyl, triphenylmethyl, methoxymethyl, ethoxymethyl,p-methoxybenzyl, p-nitrobenzyl and the like) or an acyl group (e.g.,lower (C₂₋₅) alkanoyl, benzoyl and the like), or a cyano, anN-hydroxycarbamimidoyl (preferably cyano), and specifically, cyano ispreferably used.

[0087] In the above-mentioned formula, X represents that the adjacentphenylene group and phenyl group are linked directly or via a spacer ofan atomic chain having two or less atom(s) (preferably linked directly),and the spacer of an atomic chain having two or less atom(s) may be anydivalent chain whose straight chain portion is constituted of 1 or 2atom(s). The chain may also have side chain(s). Specifically, itincludes a lower (C₁₋₄) alkylene whose straight chain portion isconstituted of 1 or 2 atoms, —CO—, —O—, —S—, —NH—, —CO—NH—, —O—CH₂—,—S—CH₂—, —CH═CH— and the like.

[0088] In the above-mentioned formula, n represents an integer of 1 or 2(preferably 1).

[0089] In the above-mentioned formula, ring A represents a benzene ringwhich may be further substituted in addition to the substituent R², andexamples of the substituent include (1) halogen (e.g., F, Cl, Br and thelike), (2) cyano, (3) nitro, (4) optionally substituted lower (C₁₋₄)alkyl, (5) lower (C₁₋₄) alkoxy, (6) an optionally substituted aminogroup (e.g., amino, N-lower (C₁₋₄) alkylamino (e.g., methylamino and thelike), N,N-dilower (C₁₋₄) alkylamino (e.g., dimethylamino and the like),N-arylamino (e.g., phenylamino and the like), alicyclic amino (e.g.,morpholino, piperidino, piperadino, N-phenylpiperadino and the like),and the like), (7) a group represented by the formula —CO—D′ [wherein D′represents a hydroxy group or lower (C₁₋₄) alkoxy in which the alkylportion may be substituted with a hydroxy group, lower (C₁₋₄) alkoxy,lower (C₂₋₆) alkanoyloxy (e.g., acetoxy, pivaloyloxy and the like),lower (C₁₋₆) alkoxycarbonyloxy (e.g., methoxycarbonyloxy,ethoxycarbonyloxy and the like) or lower (C₃₋₆) cycloalkoxycarbonyloxy(e.g., cyclohexyloxycarbonyloxy and the like)], (8) tetrazolyl, atrifluoromethanesulfonic acid amide group, a phosphoric acid group or asulfonic acid group, each of which is optionally protected withoptionally substituted lower (C₁₋₄) alkyl (including the groups similarto the “optionally substituted lower (C₁₋₄) alkyl group” exemplified asprotective groups for the group capable of forming an anion as theabove-mentioned R¹) or acyl (e.g., lower (C₂₋₅) alkanoyl, benzoyl andthe like), or the like.

[0090] One or two of these substituents may be present at thesubstitutable position(s) of the benzene ring, simultaneously, whilepreferred substituent which is further possessed by ring A in additionto the substituent R² is optionally substituted lower (C₁₋₄) alkyl(e.g., lower (C₁₋₄) alkyl optionally substituted with a hydroxy group, acarboxyl group, halogen, etc., and the like), halogen, and the like, andmore preferably, the ring A does not have any substituent except for thesubstituent R².

[0091] In the above-mentioned formula, examples of the group capable offorming an anion as R² (a group having hydrogen atom that may beliberated as a proton) include (1) a carboxyl group which may beesterified or amidated, (2) a tetrazolyl group, (3) atrifluoromethanesulfonic acid amide group (—NHSO₂CF₃), (4) a phosphoricacid group, (5) a sulfonic acid group, and the like. These groups may beprotected with an optionally substituted lower alkyl group (including agroup similar to the “optionally substituted lower (C₁₋₄) alkyl group”exemplified as protective groups for the group capable of forming ananion as the above-mentioned R¹) or an acyl group (e.g., lower (C₂₋₅)alkanoyl, benzoyl, and the like), and may be any group as long as it isa group capable of forming an anion or a group which may be convertedinto such a group under biological, i.e., physiological conditions (forexample, an in vivo reaction such as oxidation, reduction or hydrolysis,and the like with an in vivo enzyme, and the like), or chemically.

[0092] Examples of the carboxyl which may be esterified or amidated, asR², include a group represented by the formula —CO—D [wherein Drepresents (1) a hydroxy group, (2) optionally substituted amino (forexample, amino, N-lower (C₁₋₄) alkylamino, N,N-dilower (C₁₋₄) alkylaminoand the like), (3) optionally substituted alkoxy {e.g., (i) a lower(C₁₋₆) alkoxy group in which the alkyl portion is optionally substitutedwith a hydroxy group, optionally substituted amino (e.g., amino, N-lower(C₁₋₄) alkylamino, N,N-dilower (C₁₋₄) alkylamino, piperidino, morpholinoand the like), halogen, lower (C₁₋₆) alkoxy, lower (C₁₋₆) alkylthio,lower (C₃₋₈) cycloalkoxy or optionally substituted dioxolenyl (e.g.,5-methyl-2-oxo-1,3-dioxolen-4-yl and the like), or (ii) a group of theformula —O—CH(R⁶)—OCOR⁷ [wherein R⁶ represents (a) hydrogen, (b) astraight chain or branched lower alkyl group having 1-6 carbon atom(s)(e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,n-pentyl, isopentyl, neopentyl and the like), (c) a straight chain orbranched lower alkenyl group having 2-6 carbon atoms or (d) a cycloalkylgroup having 3-8 carbon atoms (e.g., cyclopentyl, cyclohexyl,cycloheptyl and the like), R⁷ represents (a) a straight chain orbranched lower alkyl group having 1-6 carbon atom(s) (e.g., methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl,n-pentyl, isopentyl, neopentyl and the like), (b) a straight chain orbranched lower alkenyl group having 2-6 carbon atoms, (c) a lower alkylgroup having 1 to 3 carbon atom(s) substituted with a cycloalkyl grouphaving 3-8 carbon atoms (e.g., cyclopentyl, cyclohexyl, cycloheptyl andthe like) or an optionally substituted aryl group (e.g., a phenyl or anaphthyl group and the like, each of which may have a halogen atom,nitro, lower (C₁₋₄) alkyl, lower (C₁₋₄) alkoxy and the like) (e.g.,benzyl, p-chlorobenzyl, phenethyl, cyclopentylmethyl, cyclohexylmethyland the like), (d) a lower alkenyl group having 2 to 3 carbon atomssubstituted with a cycloalkyl having 3-8 carbon atoms or an optionallysubstituted aryl group (e.g., phenyl or naphthyl group and the like,each of which may have a halogen atom, nitro, lower (C₁₋₄) alkyl, lower(C₁₋₄) alkoxy and the like) (e.g., a group having an alkenyl portionsuch as vinyl, propenyl, allyl, isopropenyl, and the like, for example,cinnamyl, and the like), (e) an optionally substituted aryl group (e.g.,a phenyl or naphthyl group and the like, each of which may have ahalogen atom, nitro, lower (C₁₋₄) alkyl, lower (C₁₋₄) alkoxy and thelike, such as phenyl, p-tolyl, naphthyl and the like), (f) a straightchain or branched lower alkoxy group having 1-6 carbon atom(s) (e.g.,methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy,t-butoxy, n-pentyloxy, isopentyloxy, neopentyloxy and the like), (g) astraight chain or branched lower alkenyloxy group having 2 to 8 carbonatoms (e.g., allyloxy, isobutenyloxy and the like), (h) a cycloalkyloxygroup having 3-8 carbon atoms (e.g., cyclopentyloxy, cyclohexyloxy,cycloheptyloxy and the like), (i) a lower alkoxy group having 1 to 3carbon atom(s) substituted with a cycloalkyl having 3-8 carbon atoms(e.g., cyclopentyl, cyclohexyl, cycloheptyl and the like) or anoptionaiiy substituted aryl group (e.g., a phenyl or naphthyl group andthe like, each of which may have a halogen atom, nitro, lower (C₁₋₄)alkyl, lower (C₁₋₄) alkoxy and the like) (e.g., a group having alkoxyportion(s) such as methoxy, ethoxy, n-propoxy, isopropoxy and the like,such as benzyloxy, phenethyloxy, cyclopentylmethoxy, cyclohexylmethoxyand the like), (j) a lower alkenyloxy group having 2 to 3 carbon atomssubstituted with a cycloalkyl having 3-8 carbon atoms (e.g.,cyclopentyl, cyclohexyl, cycloheptyl and the like) or an optionallysubstituted aryl group (e.g., a phenyl or naphthyl group and the like,each of which may have a halogen atom, nitro, lower (C₁₋₄) alkyl, lower(C₁₋₄) alkoxy and the like) (e.g., a group having alkenyloxy portion(s)such as vinyloxy, propenyloxy, allyloxy, isopropenyloxy and the like,such as cinnamyloxy and the like) or (k) an optionally substitutedaryloxy group (e.g., phenoxy or naphthoxy group and the like, each ofwhich may have a halogen atom, nitro, lower (C₁₋₄) alkyl, lower (C₁₋₄)alkoxy and the like, such as phenoxy, p-nitro phenoxy, naphthoxy and thelike)]}, and the like], and the like.

[0093] As R², an optionally esterified carboxyl is preferred, and thespecific examples thereof include —COOH and a salt thereof, —COOMe,—COOEt, —COOtBu, —COOPr, pivaloyloxymethoxycarbonyl,1-(cyclohexyloxycarbonyloxy)ethoxycarbonyl,5-methyl-2-oxo-1,3-dioxolen-4-ylmethoxycarbonyl, acetoxymethoxycarbonyl,propionyloxymethoxycarbonyl, n-butyryloxymethoxycarbonyl,isobutyryloxymethoxycarbonyl, 1-(ethoxycarbonyloxy)ethoxycarbonyl,1-(acetoxy)ethoxycarbonyl, 1-(isobutyryloxy)ethoxycarbonyl,cyclohexylcarbonyloxymethoxycarbonyl, benzoyloxymethoxycarbonyl,cinnamyloxycarbonyl, cyclopentylcarbonyloxymethoxycarbonyl and the like,and may be any group as long as it is a group capable of forming ananion (e.g., COO⁻, its derivative, and the like) or a group which may beconverted into such a group under biological, i.e., physiologicalconditions (for example, an in vivo reaction such as oxidation,reduction or hydrolysis, and the like with an in vivo enzyme, and thelike), or chemically, or it may be carboxyl group or a prodrug thereof.

[0094] The above-mentioned R² is preferably the group represented by theformula —CO—D [wherein D represents (1) a hydroxy group or (2) lower(C₁₋₄) alkoxy in which the alkyl portion is optionally substituted witha hydroxy group, amino, halogen, lower (C₂₋₆) alkanoyloxy (e.g.,acetoxy, pivaloyloxy and the like), lower (C₃₋₈) cycloalkanoyloxy, lower(C₁₋₆) alkoxycarbonyloxy (e.g., methoxycarbonyloxy, ethoxycarbonyloxyand the like), lower (C₃₋₈) cycloalkoxycarbonyloxy (e.g.,cyclohexyloxycarbonyloxy and the like), lower (C₁₋₄) alkoxy or lower(C₃₋₈) cycloalkoxy]. Among these, carboxyl esterified with a lower(C₁₋₄) alkyl (preferably methyl or ethyl) is preferred.

[0095] In the above-mentioned formula, examples of the “hydrocarbonresidue” of the “hydrocarbon residue which may link via a heteroatom andmay be substituted” represented by R³ include (1) an alkyl group, (2) analkenyl group, (3) an alkynyl group, (4) a cycloalkyl group, (5) an arylgroup, (6) an aralkyl group and the like. Among these, an alkyl group,an alkenyl group and a cycloalkyl group are preferred.

[0096] The alkyl group of the above-mentioned (1) may be any of straightchain or branched lower alkyl groups having about 1 to 8 carbon atom(s)such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,t-butyl, pentyl, i-pentyl, hexyl, heptyl, octyl and the like.

[0097] The alkenyl group of the above-mentioned (2) may be any ofstraight chain or branched lower alkenyl groups having 2 to 8 carbonatoms such as vinyl, propenyl, 2-butenyl, 3-butenyl, isobutenyl,2-octenyl and the like.

[0098] The alkynyl group of the above-mentioned (3) may be any ofstraight chain or branched lower alkynyl groups having 2 to 8 carbonatoms such as ethynyl, 2-propynyl, 2-butynyl, 2-pentynyl, 2-octynyl andthe like.

[0099] The cycloalkyl group of the above-mentioned (4) include lowercycloalkyl having about 3 to 6 carbon atoms such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl and the like.

[0100] Each of the above-mentioned alkyl group, alkenyl group, alkynylgroup or cycloalkyl group may be substituted with a hydroxy group, anoptionally substituted amino group (e.g., amino, N-lower (C₁₋₄)alkylamino, N,N-dilower (C₁₋₄) alkylamino and the like), halogen, alower (C₁₋₄) alkoxy group, a lower (C₁₋₄) alkylthio group and the like.

[0101] The aryl group of the above-mentioned (5) includes, for example,phenyl and the like, and the aralkyl group of the above-mentioned (6)include a phenyl-lower (C₁₋₄) alkyl and the like such as benzyl,phenethyl and the like.

[0102] Each of the above-mentioned aralkyl group or aryl group may have,at the substitutable position(s) on the benzene ring, for example,halogen (e.g., F, Cl, Br and the like), nitro, an optionally substitutedamino group (e.g., amino, N-lower (C₁₋₄) alkylamino, N,N-dilower (C₁₋₄)alkylamino and the like), lower (C₁₋₄) alkoxy (e.g., methoxy, ethoxy andthe like), lower (C₁₋₄) alkylthio (e.g., methylthio, ethylthio and thelike), lower (C₁₋₄) alkyl (e.g., methyl, ethyl and the like) and thelike.

[0103] Among the above-mentioned groups, as the “hydrocarbon residue” ofthe “hydrocarbon residue which may link via a heteroatom and may besubstituted” represented by R³, an optionally substituted alkyl oralkenyl group (e.g., lower (C₁₋₅) alkyl or lower (C₂₋₅) alkenyl groupand the like, each of which is optionally substituted with a hydroxygroup, an amino group, halogen or a lower (C₁₋₄) alkoxy group) arepreferred. Among these, a lower (C₁₋₅) alkyl (more preferably ethyl) ispreferred.

[0104] The “heteroatom” of the “hydrocarbon residue which may link via aheteroatom and may be substituted” represented by R³ include —O—,—S(O)_(m)— [m represents an integer of 0 to 2], —NR′— [R′ represents ahydrogen atom or lower (C₁₋₄) alkyl] and the like. Among these, —O— ispreferably used.

[0105] Among the above-mentioned groups, as R³, a lower (C₁₋₄) alkyl orlower (C₂₋₅) alkenyl group and the like, each of which may be linked via—O—, —S(O)_(m)— [m represents an integer of 0 to 2] or —NR′— [R′represents a hydrogen atom or lower (C₁₋₄) alkyl] and may be substitutedwith a substituent selected from a hydroxy group, an amino group,halogen and lower (C₁₋₄) alkoxy group, is preferred. Among these, alower (C₁₋₅) alkyl or a lower (C₁₋₅) alkoxy (more preferably ethoxy) ispreferred.

[0106] Among the compounds represented by the formula (I), abenzimidazol-7-carboxylic acid derivative represented by the formula(I′):

[0107] wherein R¹ is (1) a carboxyl group, (2) a tetrazolyl group or (3)a group represented by the formula:

[0108] wherein i represents —O— or —S—, j represents >=O, >=S or>=S(O)_(m), m is as defined above, ring A represents a benzene ringwhich may be further substituted with optionally substituted lower(C₁₋₄) alkyl (e.g., lower (C₁₋₄) alkyl optionally substituted with ahydroxy group, a carboxyl group, halogen and the like) or halogen andthe like, in addition to the substituent R² (preferably a benzene ringthat does not have any substituent except for R²), R² represents a grouprepresented by the formula —CO—D [wherein D represents (1) a hydroxygroup or (2) lower (C₁₋₄) alkoxy wherein the alkyl portion is optionallysubstituted with a hydroxy group, amino, halogen, lower (C₂₋₆)alkanoyloxy (e.g., acetoxy, pivaloyloxy and the like), lower (C₃₋₈)cycloalkanoyloxy, lower (C₁₋₆) alkoxycarbonyloxy (e.g.,methoxycarbonyloxy, ethoxycarbonyloxy and the like), lower (C₃₋₈)cycloalkoxycarbonyloxy (e.g., cyclohexyloxycarbonyloxy and the like),lower (C₁₋₄) alkoxy or a lower (C₃₋₈) cycloalkoxy], R³ is a lower (C₁₋₅)alkyl or lower (C₂₋₅) alkenyl group which may link via —O—, —S(O)_(m)—[m represents an integer of 0 to 2] or —NR′— [R′ represents a hydrogenatom or lower (C₁₋₄) alkyl] and may be substituted with a substituentselected from a hydroxy group, an amino group, halogen and a lower(C₁₋₄) alkoxy group (preferably lower (C₁₋₅) alkyl or lower (C₁₋₅)alkoxy; more preferably ethoxy)], or a pharmacologically acceptable saltthereof, is preferred. Among these, preferred are2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]benzimidazol-7-carboxylic acid [Candesartan],1-(cyclohexyloxycarbonyloxy)ethyl2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]benzimidazol-7-carboxylate[Candesartan cilexetil], pivaloyloxymethyl2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]benzimidazol-7-carboxylate,2-ethoxy-1-[[2′-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl]benzimidazol-7-carboxylicacid or a salt thereof and the like.

[0109] The above-mentioned benzimidazol derivatives can be synthesizedby, for example, known methods disclosed in EP 425921, EP 459136, EP553879, EP 578125, EP 520423, EP 668272 and the like, or a similarmanner thereto, and the like. Further, when Candesartan cilexetil isused, it is preferable to use the stable C type crystal disclosed in EP459136.

[0110] While the amount of the bioactive substance formulated into thesustained-release preparation of the present invention varies dependingon the kind of the bioactive substance and the like, it is generallyabout 0.1 to 50% (W/W), preferably about 0.2 to 30% (W/W), and morepreferably about 0.5 to 20% (W/W) in case of a bioactive peptide, or itis generally about 0.1 to 60% (W/W), preferably about 0.2 to 40% (W/W),and more preferably about 0.5 to 30% (W/W) in case of a non-peptidicbioactive substance.

[0111] The polymer used in the present invention is a polymer which isslightly soluble or insoluble in water and has biocompatibility.Examples thereof include polystyrene, poly-acrylic acid,poly-methacrylic acid, a copolymer of acrylic acid and methacrylic acid,nylon, tetlon, silicone polymer, dextran stearate, ethylcellulose,acetylcellulose, nitrocellulose, polyurethane, ethylene vinyl acetatecopolymer, polyvinyl acetate, polyvinyl alcohol, polyacrylicamide andthe like. Furthermore, examples of the biodegradable polymer includepolymers synthesized from one or more of α-hydroxycarboxylic acids(e.g., glycolic acid, lactic acid and the like), hydroxydicarboxylicacids (e.g., malic acid and the like), hydroxytricarboxylic acids (e.g.,citric acid and the like) etc., by catalyst-free dehydrationpolycondensation, which have free carboxyl group(s), or a mixturethereof, poly-α-cyanoacrylic esters, polyamino acids (e.g.,poly-y-benzyl-L-glutamic acid and the like), maleic anhydride polymers(e.g., a styrene/maleic acid polymer and the like), and the like. Thepolymer may be a homopolymer or a copolymer. The type of polymerizationmay be of random, block or graft. When the above-mentionedα-hydroxycarboxylic acids, hydroxydicarboxylic acids andhydroxytricarboxylic acids have optically active centers in theirmolecular structures, they may be any of the D-, L- andDL-configurations.

[0112] Among these polymers, the biodegradable polymer having a freeterminal carboxyl group such as a polymer synthesized fromα-hydroxycarboxylic acids (e.g., glycolic acid, lactic acid and thelike) (e.g., poly-lactic acid, lactic acid/glycolic acid copolymer, andthe like), poly-α-cyanoacrylic acid esters and the like are preferred.

[0113] The biodegradable polymer is more preferably a polymersynthesized from α-hydroxycarboxylic acids and the like, especiallypreferably a lactic acid/glycolic acid polymer and the like.

[0114] Not only homopolymers such as poly-lactic acid, poly-glycolicacid, etc., but also lactic acid/glycolic acid copolymers are sometimessimply referred to as the lactic acid/glycolic acid polymer hereininclusively.

[0115] When the lactic acid/glycolic acid polymer (a lacticacid/glycolic acid copolymer or homopolymer) is used as thebiodegradable polymer, its composition ratio (mol %) is preferably about100/0 to about 40/60, more preferably about 85/15 to about 50/50.

[0116] The weight-average molecular weight of the above-described lacticacid/glycolic acid polymer is preferably about 3,000 to about 50,000,more preferably about 3,000 to about 25,000, further more preferablyabout 5,000 to about 20,000.

[0117] The degree of dispersion (weight-average molecularweight/number-average molecular weight) of the lactic acid/glycolic acidpolymer is preferably about 1.2 to about 4.0, more preferably about 1.5to about 3.5.

[0118] Regarding the weight-average molecular weight and the degree ofdispersion used herein, the former is a value converted into polystyreneas determined by gel permeation chromatography (GPC) using as referencesubstances 9 kinds of polystyrenes having the weight-average molecularweights of 120,000, 52,000, 22,000, 9,200, 5,050, 2,950, 1,050, 580 and162, respectively, and the latter is calculated therefrom. The abovedetermination is carried out using GPC column KF804L ×2 (manufactured byShowa Denko K. K.) and RI monitor L-3300 (manufactured by Hitachi Ltd.)with chloroform as a mobile phase.

[0119] Further, the biodegradable polymer having a free terminalcarboxyl group is a biodegradable polymer in which the number-averagemolecular weight based on GPC measurement and the number-averagemolecular weight based on terminal group quantification almost agreewith each other. The number-average molecular weight based on terminalgroup quantification is calculated as follows:

[0120] About 1 to 3 g of the biodegradable polymer is dissolved in amixed solvent of acetone (25 ml) and methanol (5 ml), and the solutionis quickly titrated with a 0.05 N alcoholic solution of potassiumhydroxide using phenolphthalein as an indicator while stirring at roomtemperature (20° C.) to determine the carboxyl group content; thenumber-average molecular weight based on terminal group quantificationis calculated from the following equation: Number-average molecularweight based on terminal group quantification=20000 ×A/B

[0121] A: the mass (g) of the biodegradable polymer

[0122] B: the amount (ml) of 0.05 N alcoholic solution of potassiumhydroxide added until the titration end point

[0123] The number-average molecular weight based on terminal groupquantification is an absolute value, while the number-average molecularweight based on GPC measurement is a relative value that variesdepending on various analytical conditions (e.g., kind of mobile phase,kind of column, reference substance, slice width chosen, baselinechosen, etc.). Therefore, although it is difficult to expressunequivocally and numerically, such description that “the number-averagemolecular weight based on GPC measurement and that based on terminalgroup quantification almost agree with each other” means, for example,that the number-average molecular weight based on terminal groupquantification of a polymer synthesized from α-hydroxycarboxylic acidsfalls within the range from about 0.5 to about 2 times, preferably fromabout 0.7 to about 1.5 times as much as the number-average molecularweight based on GPC measurement.

[0124] For example, in case of a polymer having a free terminal carboxylgroup and synthesized from one or more α-hydroxycarboxylic acids bycatalyst-free dehydration polycondensation, the number-average molecularweight based on GPC measurement and the number-average molecular weightbased on terminal group quantification almost agree with each other. Onthe other hand, in case of a polymer having substantially no freeterminal carboxyl group and synthesized from a cyclic dimer byring-opening polymerization using a catalyst, the number-averagemolecular weight based on terminal group quantification is significantlyhigher than (more than about 2 times) that based on GPC measurement.This difference makes it possible to clearly distinguish a polymerhaving a free terminal carboxyl group from a polymer having no freeterminal carboxyl group.

[0125] The lactic acid/glycolic acid polymer having a free terminalcarboxyl group can be produced by a per se known process, for example,that described in JP 61-28521 A (e.g., a process by a catalyst-freedehydration polycondensation reaction, or a dehydration polycondensationreaction in the presence of an inorganic solid acid catalyst, etc.).

[0126] While the decomposition/disappearance rate of the lacticacid/glycolic acid polymer varies widely, depending on the compositionratio or the weight-average molecular weight, the release duration canbe extended (e.g., for about 6 months) by lowering the ratio of glycolicacid or increasing the molecular weight, sincedecomposition/disappearance is generally delayed as the ratio ofglycolic acid decreases. In contrast, the release duration can beshortened (e.g., for about one week) by increasing the ratio of glycolicacid or decreasing the molecular weight. For obtaining a one week to twomonths type sustained-release preparation, it is preferable to use thelactic acid/glycolic acid polymer whose composition ratio andweight-average molecular weight are within the above-described ranges.

[0127] Therefore, the composition of the biodegradable polymer used inthe present invention is preferably selected according to the desiredkind of a bioactive peptide and the desired duration ofsustained-release. Specifically, for example, when GH is used as thebioactive peptide, a lactic acid/glycolic acid polymer is preferablyused. As the lactic acid/glycolic acid polymer, a preferred polymer is alactic acid/glycolic acid copolymer having a lactic acid/glycolic acidcomposition ratio (mol %) of about 85/15 to about 50/50, more preferablyabout 75/25 to about 50/50. The weight-average molecular weight thereofis preferably about 8,000 to about 20,000, more preferably about 10,000to about 20,000. Further, the degree of dispersion (weight-averagemolecular weight/number-average molecular weight) of the lacticacid/glycolic acid polymer is about 1.2 to about 4.0, more preferablyabout 1.5 to about 3.5.

[0128] The lactic acid/glycolic acid polymer used can be produced byknown methods such as those described in the above publications and thelike. The polymer is preferably that produced by catalyst-freedehydration polycondensation. Any organic solvent used for theproduction of the polymer and remaining in the polymer is removed afterthe polymerization. As a method for this purpose, there are, forexample, heat drying, vacuum drying, flash drying with dried gas, etc.However, such a solvent can be much more quickly removed by contactingwith continuously provided high-pressure gas according to the presentinvention, thereby significantly reducing the time required for removingthe solvent. It is preferable to use the lactic acid/glycolic acidpolymer (PLGA) wherein the number-average molecular weight based onterminal group quantification and the number-average molecular weightbased on GPC measurement almost agree with each other.

[0129] Further, two kinds of lactic acid/glycolic acid polymersdifferent in the composition ratio and/or the weight-average molecularweight may be used by mixing them in an arbitrary ratio. An examplethereof is a mixture of a lactic acid/glycolic acid copolymer having thecomposition ratio of lactic acid/glycolic acid (mol %) of about 75/25and the weight-average molecular weight of about 10,000, and a lacticacid/glycolic acid copolymer having the composition ratio of lacticacid/glycolic acid (mol %) of about 50/50 and the weight-averagemolecular weight is about 12,000. The preferred weight ratio of thesecopolymers upon mixing is about 25/75 to about 75/25.

[0130] The biodegradable polymer used in the present invention may be ametal salt of the above mentioned biodegradable polymer. For example,there can be used the various polyvalent metal salts of thebiodegradable polymer described in WO97/01331, and the like. Preferably,a polyvalent metal salt of the lactic acid/glycolic acid polymer and thelike, (more preferably, zinc salt, calcium salt, magnesium salt and thelike, further more preferably zinc salt and the like) can be used. Thespecies of the metal of the polyvalent metal salt is not specificallylimited as long as it does not cause any adverse effect to a livingbody. For example, there can be used a polyvalent metal such as adivalent metal (e.g., iron, zinc, copper, calcium, magnesium, aluminum,tin, manganese and the like), a trivalent metal (e.g., iron, aluminum,manganese and the like), a tetravalent metal (e.g., tin and the like)and the like.

[0131] A metal salt of the biodegradable polymer is sometimes referredto as the biodegradable polymer herein inclusively. For example, in caseof a polyvalent metal salt of the lactic acid/glycolic acid polymer,sometimes, it is also referred to as the lactic acid/glycolic acidpolymer.

[0132] The above polyvalent metal salt of the biodegradable polymer canbe produced by the method described in WO97/01331 or similar methods.

[0133] In case that a polyvalent metal salt of the biodegradable polymeris a zinc salt, it can be produced by reacting the biodegradable polymerwith zinc oxide in an organic solvent.

[0134] In this method, first, a solution of the biodegradablepolymer-zinc oxide complex in an organic solvent is prepared bycoexistence of the biodegradable polymer with zinc oxide in the organicsolvent. In that case, although the concentration of the biodegradablepolymer in the solvent varies depending on the molecular weight thereof,a kind of the organic solvent and the like, for example, theconcentration is about 0.1 to about 80% (W/W), preferably about 1 toabout 70% (W/W), more preferably about 2 to about 60% (W/W). Further,although the amount of zinc oxide to be added varies depending on thekind of the organic solvent, for example, when the desired bioactivesubstance is a peptide, the amount is about 0.001 to about 2% (W/W),preferably about 0.01 to about 1.5% (W/W), more preferably about 0.1 toabout 1% (W/W), when the desired bioactive substance is a non-peptide,the amount is about 0.001 to about 30% (W/W), preferably about 0.01 toabout 20% (W/W), more preferably about 0.1 to about 10% (W/W), based onthe amount of the biodegradable polymer, as described in JP 10-231252 A.

[0135] Regarding the order of the addition of the biodegradable polymerand zinc oxide to the organic solvent, zinc oxide in the form of apowder or suspended in the organic solvent can be added to a solutionprepared by dissolving the biodegradable polymer in the organic solvent,or on the contrary, a solution of the biodegradable polymer in theorganic solvent can be added to a suspension prepared by suspending zincoxide in the organic solvent. Further, both of the biodegradable polymerand zinc oxide can be mixed in the form of powders, then the organicsolvent can be added thereto. When the desired bioactive substance is anon-peptide, the organic solvent can be added after mixing of thebiodegradable polymer, zinc oxide and the bioactive substance in theform of powders.

[0136] The content of the biodegradable polymer in the preparation ofthe present invention is generally about 30 to 99.9% (W/W), preferablyabout 60 to 97% (W/W), and more preferably about 70 to 90% (W/W).

[0137] The preparation of the present invention is produced by forming asolid material containing the bioactive substance and the biodegradablepolymer and contacting the solid material with high-pressure gas.

[0138] The solid material containing the bioactive substance and thebiodegradable polymer is formed by, when the bioactive substance is abioactive peptide, for example, removing a solvent from a S/O dispersionobtained by dispersing a powder (S phase), which has been obtained bylyophilizing a solution of the bioactive peptide, in a solution of thebiodegradable polymer in an organic solvent (O phase), or removing asolvent from a W/O emulsion obtained by dispersing an aqueous phase (Wphase), which is an aqueous solution of the bioactive peptide, in asolution of the biodegradable polymer dissolved in an organic solvent,or removing a solvent from a solution of both bioactive peptide andbiodegradable polymer dissolved in an organic solvent (O phase). As amethod for this, there are, for example, (a) in-water drying method(S/O/W method and W/O/W or O/W method), (b) phase separation method(coacervation method) and (c) spray-drying method, or similar methodsthereto and the like. In the present description, the solid materialmeans a material in which constituents are linked to each otherphysically or chemically. The solid material includes, but notspecifically limited to, microcapsules and the like.

[0139] The organic solvent used for dissolving the biodegradable polymerpreferably has the boiling point of not lower than 30° C. Examples ofthe organic solvent includes halogenated hydrocarbons such asdichloromethane, dichloroethane, chloroform, carbon tetrachloride andthe like, aliphatic hydrocarbons such as pentane, hexane, heptane,cyclohexane, petroleum benzine, petroleum ether and the like, aromatichydrocarbons such as toluene, xylene and the like, alcohols such asmethyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol,benzyl alcohol and the like, polyhydric alcohols such as ethyleneglycol, propylene glycol and the like, esters such as methyl acetate,ethyl acetate and the like, organic acids such as formic acid, aceticacid, trifluoroacetic acid, trichloroacetic acid and the like, etherssuch as diethyl ether, isopropyl ether, dioxane, tetrahydrofuran and thelike, ketones such as acetone, methyl ethyl ketone and the like,nitrogen-containing compounds such as acetonitrile, propionitrile,pyridine, dimethylacetamide, dimethylformamide and the like,sulfur-containing compounds such as dimethylsulfoxide and the like, andthe like. These may be mixed in a suitable ratio. A solvent contained ina substance, especially an organic compound, e.g., a medicament shouldbe substantially removed from a product in view of properties of themedicament. Furthermore, for foods and general chemicals, residualsolvents in products are strictly regulated depending on theirapplication. The allowable amount of a residual solvent for medicamentsis described in the guideline based on the ICH (“A guideline forresidual solvent in medicament”, Pharm. Tech. Japan 16(5), 687-704,2000), and for example, the concentration limit for dichloromethane(classified into the Class 2) is 600 ppm, and the concentration limitfor acetone (classified into the Class 3) is 0.5% (5,000 ppm).

[0140] Moreover, an organic solvent used in dissolution of thebiodegradable polymer preferably has a boiling point of not higher than120° C. The organic solvent includes such as halogenated hydrocarbons(e.g., dichloromethane, chloroform and the like), alcohols (e.g.,ethanol, methanol and the like), ethyl acetate, acetonitrile and thelike. These may be mixed in a suitable ratio. When an organic solvent isused alone, for example, dichloromethane, ethyl acetate, acetonitrileand the like are preferred. When organic solvents are used as a mixedsolvent, for example, a combination of halogenated hydrocarbons (e.g.,dichloromethane, chloroform and the like) and alcohols (e.g., ethanol,methanol and the like) or acetonitrile is preferred. The mixing ratio(volume ratio) of the halogenated hydrocarbons and alcohols oracetonitrile is about 100:1 to about 1:1, and it is desirable to use amixed solvent having a mixing ratio of preferably about 30:1 to about2:1. Furthermore, while the concentration of the biodegradable polymerin a solution varies depending on the molecular weight, the kind of anorganic solvent, and the like, for example, it is about 0.01 to about80% (W/W), preferably about 0.1 to about 70% (W/W), and more preferablyabout 1 to about 60% (W/W).

[0141] Hereinafter, a method for microcapsulation, in case of theproduction of sustained-release microcapsules as the preparation andusing a bioactive peptide as the bioactive substance, will be explainedin detail.

[0142] (a-1) In-Water Drying Method (S/O/W Method)

[0143] According to this method, first, a water-miscible organic solventand/or a volatile salt are added to an aqueous solution of the bioactivepeptide, and then, a bioactive peptide powder (S phase) is produced bylyophilization. A biodegradable polymer is dissolved in an organicsolvent, and then, the above bioactive peptide powder is dispersed intothe resulting organic solvent solution. The ratio (ratio by weight) ofthe bioactive peptide and the biodegradable polymer is, for example,about 1:1000 to about 1:1, preferably about 1:200 to about 1:5, morepreferably about 1:100 to about 1:5. Preferably, an external physicalenergy is applied to disperse the bioactive peptide powder uniformly inthe organic solvent solution. As a method for this, there can be used,for example, irradiation of ultrasonic wave, a turbine stirrer, ahomogenizer and the like. As to the average particle size of thebioactive peptide in the organic solvent solution, it is preferably notmore than about 10 μm, more preferably about 0.1 to 10 μm, further morepreferably about 0.5 to 5 μm. In the present invention, the averageparticle size of the bioactive peptide means the value obtained by usinga laser analytic particle size distribution measuring device (SALD2000A,manufactured by Shimadzu Corporation) after dispersing the bioactivepeptide in an organic solvent such as dichloromethane by using ahomogenizer. In this process, the bioactive peptide is added to theorganic solvent at the concentration of about 20 to 100 mg/ml, and thendispersed using a homogenizer, such as Polytron (manufactured byKinematica) at about 20,000 rpm for about 30 seconds to 1 minutes. Thedispersion is diluted appropriately with the organic solvent so that theaverage particle size can be measured with the above particle sizedistribution measuring device, followed by analysis.

[0144] Then, the organic solvent dispersion (S/O dispersion) thusprepared is added to an aqueous solvent (W phase), and then the sameexternal physical energy as that mentioned above, for example,irradiation of ultrasonic wave, a turbine stirrer, a homogenizer and thelike is applied to form a S/O/W emulsion. Then, the organic solvent of Ophase is evaporated to produce microcapsules. At this time, the volumeof the aqueous phase is generally selected from about 1 times to about10,000 times, preferably about 2 times to about 5,000 times, morepreferably about 5 times to about 2,000 times as much as the volume ofthe O phase.

[0145] An emulsifier can be added to the above external aqueous phase.As the emulsifier, there can be used any one which is generally capableof forming a stable S/O/W emulsion. Examples of the emulsifier includeanionic surfactants, nonionic surfactants, polyoxyethylene castor oilderivative, polyvinylpyrrolidones, polyvinyl alcohols,carboxymethylcelluloses, lecithin, gelatin, hyaluronic acids and thelike. These emulsifiers can be used by appropriately combining them. Theconcentration of the emulsifier in the external aqueous phase is,preferably about 0.001% to 20% (w/w), more preferably about 0.01% to 10%(w/w), particularly preferably about 0.05% to 5% (w/w).

[0146] The thus-obtained microcapsules are recovered by centrifugationor filtration, washed with distilled water to remove the emulsifier andthe like adhering to the surface of microcapsules, re-dispersed indistilled water, and lyophilized.

[0147] In the present invention, examples of the water-miscible organicsolvent, which can be added to the aqueous solution of the bioactivepeptide, include alcohols (e.g. methanol, ethanol, isopropanol and thelike, preferably methanol, ethanol and the like), acetone and the like.These may be used by mixing them at an appropriate ratio. Preferably, analcohol, more preferably ethanol is used alone. The amount(concentration) to be added to the aqueous solution of the bioactivepeptide is about 0.03 to 0.5% (V/V), preferably about 0.06 to 0.25%(V/V); more preferably about 0.1 to 0.15% (V/V), in terms ofvolume-ratio. By further lyophilizing the resultant aqueous solution ofthe bioactive peptide obtained by addition of the water-miscible organicsolvent, it is possible to prepare a bioactive peptide powder which iseasy to handle (superior operability) and is very fine (a small particlesize).

[0148] In the present invention, as the volatile salt, which is added tothe aqueous solution of the bioactive peptide, there are, for example,ammonium salts (e.g., ammonium acetate, ammonium bicarbonate, ammoniumcarbonate, ammonium chloride and the like, preferably ammonium acetateand the like). The volatile salt can be used by mixing them at anappropriate ratio. The added amount of the volatile salt is about 10times to about 80 times mole, preferably about 10 times to about 70times mole, more preferably about 15 times to about 70 times mole,further more preferably about 20 times to about 70 times mole, mostpreferably about 20 times to about 50 times mole as much as the aqueoussolution of the bioactive peptide in terms of mole ratio. Bylyophilizing the resultant aqueous solution of the bioactive peptideobtained by addition of the volatile salt in a similar manner as theaddition of the water-miscible organic solvent, it is possible toprepare the bioactive peptide powder which is easy to handle (superioroperability) and is very fine (a small particle size).

[0149] In the present invention, the water-miscible organic solventand/or the volatile salt added to the aqueous solution of the bioactivepeptide can be used alone or in appropriate combination thereof. Whenthe water-miscible organic solvent and the volatile salt are used incombination thereof, they can be added to the aqueous solution of thebioactive peptide in accordance with the above amounts respectively.

[0150] (a-2) In-water Drying Method (W/O/W Method)

[0151] According to this method, water or a suitable buffer is added tothe bioactive peptide to give a solution of the bioactive peptide (Wphase). The biodegradable polymer is then dissolved in an organicsolvent, and to this organic solvent solution is added theabove-mentioned solution of the bioactive peptide and the mixture isdispersed. The thus-obtained W/O emulsion is added to an aqueous solvent(W phase). According to the same method as the above-mentioned S/O/Wmethod, microcapsules are obtained through a W/O/W emulsion.

[0152] (a-3) In-water Drying Method (O/W Method)

[0153] According to this method, the biodegradable polymer together withthe bioactive peptide are dissolved in an organic solvent. The organicsolvent (O phase) is then added to an aqueous solvent (W phase).According to the same method as the above-mentioned S/O/W method,microcapsules are obtained through an O/W emulsion.

[0154] (b) Phase Separation Method (Coacervation Method)

[0155] According to this method, a coacervating agent is gradually addedto the S/O dispersion of (a-1) or the W/O emulsion of (a-2) or the Ophase solution of (a-3) as described above with stirring to precipitateand solidify microcapsules. The amount of the coacervating agent to beadded is about 0.01 to about 1,000 times by volume, preferably about0.05 to about 500 times by volume, especially preferably about 0.1 toabout 200 times by volume as much as the volume of the above dispersion.Any coacervating agent can be used, as long as it is a polymeric,mineral oil or vegetable oil compound which is miscible with the organicsolvent used for dissolution of the biodegradable polymer but does notdissolve the biodegradable polymer used. Specifically, examples of thecoacervating agent include silicone oil, sesame oil, soybean oil, cornoil, cottonseed oil, coconut oil, linseed oil, mineral oil, n-hexane,n-heptane and the like. Two or more of these can be used in combination.The thus-obtained microcapsules are recovered by filtration, washedrepeatedly with heptane and the like to remove the coacervating agent.Further, washing is carried out in the same manner as that in the above(a), followed by lyophilization.

[0156] In the production of microcapsules by the in-water drying methodor coacervation method, an antiaggregation agent can be added forpreventing aggregation of particles. Examples of the antiaggregationagent can be used, for example, water-soluble polysaccharides such asmannitol, lactose, glucose, starches (e.g., corn starch and the like),hyaluronic acid and its alakaline metal salt, etc.; protein such asglycine, fibrin, collagen, etc.; inorganic salts such as sodiumchloride, sodium hydrogen phosphate, etc.; and the like.

[0157] (c) Spray-Drying Method

[0158] In this method, microcapsules are produced by spraying the S/Odispersion of (a-1), the W/O emulsion of (a-2) or the O phase solutionof (a-3) described above via a nozzle into the drying chamber of a spraydrier to volatilize the organic solvent in fine droplets within a veryshort time. As the nozzle, there are, for example, a two-fluid nozzletype, a pressure nozzle type and a rotary disc type and the like. It isalso advantageous, if necessary, to spray an aqueous solution of theabove-described antiaggregation agent via another nozzle in order toprevent aggregation of microcapsule particles.

[0159] The solid material formed by the above-mentioned method such asmicrocapsules containing the bioactive substance and the biodegradablepolymer, and the like, is then contacted with high-pressure gas(preferably carbon dioxide) to further extract and remove the organicsolvent.

[0160] Specifically, for example, a lyophilized microcapsule powderobtained by (a) is fed into an extraction vessel, and extractiontreatment is carried out with an extraction system comprising a carbondioxide delivery pump and a pressure regulating valve. Alternatively, amicrocapsule suspension before lyophilization, obtained by (a) or (b)may be fed into an extraction vessel and subjected to extractiontreatment similarly. In these cases, the extraction treatment isdesirably carried out under more gentle conditions so as to notdeteriorate the quality of a sustained-release preparation.

[0161] The high-pressure gas in the present invention is gas at pressurenot less than the atmospheric pressure at a given temperature but notmore than the liquefying pressure at said temperature.

[0162] Examples of the high-pressure gas used in the present inventioninclude carbon dioxide, nitrous oxide, nitrogen, helium, argon, alkane(e.g., ethane, propane and the like), alkene (e.g., ethylene and thelike), and the like. While these may be used by mixing them in asuitable ratio, preferably, it is desirable to use carbon dioxide alone.

[0163] When a temperature of high-pressure gas contacting with apreparation is much higher than the glass transition temperature of abiodegradable polymer used as a substrate of the preparation, the riskof adhesion, deformation, decomposition of the bioactive substance,deterioration and the like of the preparation increases. The glasstransition temperature in the present invention means medium glasstransition temperature obtained by rising temperature at the rate of 10or 20° C./min using a differential scanning calorimeter (DSC).Alternatively, when a temperature of high-pressure gas is too low, theremoval of an organic solvent becomes insufficient. The organic solventis preferably removed to less than 1,000 ppm, preferably less than 500ppm, and more preferably less than 100 ppm. Therefore, the advantageoustemperature for using carbon dioxide as high-pressure gas in the presentinvention is within a temperature range of +20 to −60° C., preferably+10 to −50° C., more preferably 0 to −40° C., still more preferably −5to -30° C., and the most preferably −10 to −25° C., based on the glasstransition temperature of the biodegradable polymer (generally about 20to 60° C.).

[0164] While the range of pressure varies depending on the selectedhigh-pressure gas, but generally, when the pressure of high-pressure gasis too high, the risk of adhesion, deformation, increase of the initialrelease immediately after administration and the like for themicrocapsules increases, or when the pressure is too low, the removal ofthe organic solvent becomes insufficient. The advantageous pressure forusing carbon dioxide as high-pressure gas in the present inventionpressure is about 1 to 7 MPa, preferably about 1 to 4 MPa, and morepreferably about 2 to 4 MPa.

[0165] While the period for contacting with the high-pressure gas variesdepending on the pressure of the high-pressure gas, temperature, theamount of microcapsules to be treated and the like, it is preferablyabout 5 minutes to about 48 hours when carbon dioxide is used ashigh-pressure gas. More preferably, it is about 10 minutes to about 12hours.

[0166] Hereinafter the step for high-pressure gas treatment ofmicrocapsules using carbon dioxide in a high-pressure gaseous state willbe explained in more detail with referring to FIG. 1. FIG. 1 is aschematic drawing, which exemplifies an apparatus used for thehigh-pressure gas treatment in the present invention. Such apparatus forhigh-pressure gas treatment comprises, for example, as shown in FIG. 1,a liquefied carbon dioxide bomb 1, a carbon dioxide delivery pump 2, aheat exchanger 3, an extraction vessel 4, a thermostat 5, a detector 6,an automatic pressure-regulating valve 7 and a recovery vessel 8.Microcapsules to be treated are fed into the extraction vessel 4, andthe apparatus is sealed and heated to a predetermined temperature. Theliquefied carbon dioxide is then delivered from the liquefied carbondioxide bomb 1 to the heat exchanger 3 by the carbon dioxide deliverypump 2, heated to a predetermined temperature, and converted into ahigh-pressure gaseous state. The carbon dioxide in the high-pressuregaseous state is then blown into the extraction vessel 4 to dissolve andextract the solvent in the microcapsules into the high-pressure gas. Theextracted solvent is recovered in the recovery vessel 8 via the detector6 and automatic pressure regulating valve 7. The pressure applied to thewhole system is controlled by the automatic pressure regulating valve 7connected to the lowest downstream. By contacting with the high-pressuregas for a given period, the excess amount of the initial release of thebioactive substance immediately after administration is markedlysuppressed, and the residual organic solvent can be removed withoutproducing aggregates, related substances or reactants of the bioactivepeptide.

[0167] The sustained-release preparation of the present invention ispreferably in the form of fine particles. That is, the sustained-releasepreparation does not provide undue pain to a patient, when it isadministered to said patient using an injection needle, which isgenerally used for subcutaneous or intramuscular injection. The particlesize of the sustained-release preparation is, for example, about 0.1 to300 μm, preferably about 1 to 150 μm, specifically preferably about 2 to100 μm in terms of a mean particle diameter. The content of thebioactive substance contained in the sustained-release preparation ofthe present invention is, for example, in case of a bioactive peptide,generally about 0.1 to 50% (W/W), preferably about 0.2 to 30% (W/W), andmore preferably about 0.5 to 20% (W/W). The content of the biodegradablepolymer contained in the sustained-release preparation of the presentinvention is generally about 30 to 99.9% (W/W), preferably about 60 to97% (W/W), and more preferably about 70 to 90% (W/W).

[0168] The initial release percentage of the sustained-releasepreparation of the present invention [the release percentage up to oneday (24 hours) after administration] is, in case of a bioactive peptide,preferably about not more than 40%, more preferably about 1 to 40%, andmore preferably about 3 to 35%.

[0169] The sustained-release preparation of the present invention can beadministered as microcapsules or as preparations in various formsprepared by using microcapsules as a raw material, such as parenteralpreparations (e.g., injectable preparations or preparations forimplantation in muscle, subcutaneous, organs and the like, preparationsfor administering to mucosa onto cavitas nasi, rectum, uterus, etc.),oral preparations (e.g., capsules (hard capsules, soft capsules, etc.),solid preparations such as granules and powders, etc., liquidpreparations such as suspensions, etc.), and the like.

[0170] In particular, the sustained-release preparation of the presentinvention is preferably for injection. For example, in case that thesustained-release preparation is microcapsules, it is possible to obtaina practical sustained-release preparation for injection by formulatingthe microcapsules in an aqueous suspension together with a dispersingagent (e.g., a surfactant such as Tween 80, HCO-60, etc.,polysaccharides such as carboxymethyl celluloses, sodium alginate,hyaluronic acid, etc.), a preservative (e.g., methylparaben,propylparaben, etc.), a tonicity agent (e.g., sodium chloride, mannitol,sorbitol, glucose, etc.), and the like. It is also possible to obtain apractical sustained-release preparation for injection by dispersing themicrocapsules together with a vegetable oil such as sesame oil, cornoil, etc., or a mixture thereof with a phospholipid such as lecithin, ora medium-chain fatty acid triglyceride (e.g., Miglyol 812) to prepare anoily suspension.

[0171] When the sustained-release preparation is, for example,microcapsules, the particle size of the sustained-release preparationfor an injectable suspension can be selected from the range satisfyingthe requirements for the degree of dispersion and the needle passabilityfor the injection. For example, the particle size is within the range ofabout 0.1 to about 300 μm, preferably about 1 to about 150 μm, morepreferably about 2 to about 100 μm, as the average particle size.

[0172] Methods for producing a sterile preparation from the abovemicrocapsules include, but are not limited to, to carry out entireproduction steps aseptically, to sterilize with gamma rays, to add anantiseptic, and the like.

[0173] The sustained-release preparation can be safely used in mammals(e.g., human, cattle, pig, dog, cat, mouse, rat, rabbit and the like)with low toxicity.

[0174] Indication of the sustained-release preparation varies dependingon a bioactive peptide used. The sustained-release preparation is usefulto prevent or treat diabetes when insulin is used as the bioactivepeptide; viral hepatitis (e.g., type C hepatitis, HBe antigen-positiveactive hepatitis and the like) and cancer (e.g., renal carcinoma,multiple myeloma and the like) when interferon-α is used; anemia (e.g.,anemia during dialysis of kidney and the like) when erythropoietin isused; neutropenia (e.g., in cancer therapy and the like) and infectionswhen G-CSF is used; cancer (e.g., hemangioendothelioma and the like)when IL-2 is used; fracture, wound (e.g., bedsore and the like),periodontitis and gastrointestinal ulcer when FGF is used;thrombocytopenia when FGF-9 is used; senile dementia and neuropathy whenNGF is used; thrombosis when TPA is used; and cancer when tumor necrosisfactor is used. Further, the sustained-release preparation containing GHis applied to Turner's syndrome, chronic renal diseases, achondroplasia,and adult hypopituitarisin (adult GHD), in addition to pituitarydwarfism, based on growth hormone activity of GH. Further, since, GH isreported to be applied to diseases such as Down syndrome, Silversyndrome, hypochondroplasia and juvenile chronic arthritis to provideexcellent therapeutic effects, the sustained-release preparationcontaining GH can also be applied to these diseases. Thesustained-release preparation containing GH is also useful to prevent ortreat congestive heart-failure and the like. The other indications towhich the sustained-release preparation containing GH can be appliedinclude, hematogenesis during organ implantation or treatment for apatient suffering from AIDS with a drug, improvement ofhypoalimentation, renal anemia, angina pectoris, hyperlipidemia,obesity, acceleration of treatment for burn, wound or ulcer,invasiveness from surgery (operation, lesion), early recovery afteroperation, sepsis, prevention of fracture due to osteoporosis, earlyrecovery of muscular strength of a patient suffering from fracture dueto osteoporosis, amyotropic lateral scelosis (ALS), decubitus and thelike. Furthermore, it is expected to have effects as an antiaging agentaiming at improving the quality of life (QOL) for frail aged persons, oreffects for suppressing the development or improving neurodegenerativediseases (Alzheimer's disease, Parkinson's disease, cerebrovasculardisease and the like) due to the nerve protective effect of hGH. Byforming GH into a sustained-release preparation, drug effects superiorto those of a GH subcutaneous injection can be obtained for theseindications. When the bioactive substance is candesartan, thepreparation is effective for the prevention or improvement ofcardiomegaly, cardiac failure, myocardial infarct, cerebral stroke,ischemic peripheral neuropathy, myocardial ischemia, venousincompetence, development of cardiac failure after myocardial infarct,diabetic nephropathy, nephritis, glomerular nephritis, arteriosclerosis,vascular hypertrophy, vascular hypertrophy or occulusion afterpercutaneous transluminal coronary angioplasty, vascular re-occulusionafter bypass operation, hyperaldosteronism, glomerular sclerosis, renalfailure, glaucoma, ocular hypertension, hyperlipidemia, stenocardia,aneurysm, coronary arteriosclerosis, cerebral arteriosclerosis,peripheral arteriosclerosis, thrombosis, central nerve system disease,Alzheimer's disease, amnesia, depression, amnestic syndrome, seniledementia, dysesthesia, multiple organ failure, prevention or treatmentof disease or sclerodermia associated with endothelial disorder, orsymptom of anxiety, symptom of strain, unpleasantmental state ormaldigestion.

[0175] Although the dose of the sustained-release preparation variesdepending on a particular kind and amount of the bioactive peptide,release duration, target disease, subject animal species and otherfactors, it can be set at any level, as long as an effectiveconcentration of the bioactive peptide in the body is maintained. Forexample, when the sustained-release preparation is one designed for twoweek release, the dose of the bioactive peptide can be suitably chosenfrom the range of preferably about 0.0001 to about 10 mg/kg body weight,more preferably about 0.05 to about 3 mg/kg body weight, per an adult.The preferred administration frequency of the sustained-releasepreparation can be suitably chosen from once a week, once every twoweeks, once a month, once every two months and the like, depending on aparticular kind and amount of the bioactive peptide, dosage form,release duration, target disease, subject animal species and otherfactors. Preferably, the sustained-release preparation includes a oneweek to two months type sustained-release preparation, more preferablyone week to one month type sustained-release preparation.

[0176] When the bioactive peptide as an active component in thesustained-release preparation is, for example, insulin, the dose peradministration to an diabetic adult is suitably chosen from the range ofusually about 0.001 to about 1 mg/kg body weight, preferably about 0.01to about 0.2 mg/kg body weight, as an effective ingredient. And thepreferred administration frequency is once a week.

[0177] When the bioactive peptide as an active component in thesustained-release preparation is GH, the dose can be set at any level,as long as an effective concentration of GH in the body is maintained,although varying depending on a particular kind and amount of GH,release duration, target disease, subject animal species and otherfactors. Regarding the treatment of the above described diseases, whenthe sustained-release preparation is one designed for two week release,the dose of GH can be suitably chosen from the range of about 0.01 toabout 5 mg/kg body weight (about 0.03 to about 15 IU/kg body weight),more preferably about 0.05 to about 1 mg/kg body weight (about 0.15 toabout 3 IU/kg body weight), per a child or an adult for safeadministration. The preferred administration frequency can be suitablychosen from once a week, once every two weeks, once a month and etc.,depending on a particular amount of GH, dosage form, release duration,target disease, subject animal species and other factors, preferably oneweek to two months-type sustained-release preparation, more preferablyone week to one month-type sustained-release preparation.

[0178] The sustained-release preparation is preferably stored atordinary temperature or in a cold place. More preferably, thesustained-release preparation is stored in a cold place. The “ordinarytemperature” and the “cold place” are defined in the Pharmacopoeia ofJapan. Namely, the “ordinary temperature” means 15 to 25° C., and the“cold place” means a temperature of not more than 15° C. In the “coldplace”, it is more preferably about 2 to 8° C.

[0179] Hereinafter the present invention will be explained morespecifically with referring to the Reference Examples, Examples and TestExamples, which do not limit the present invention.

REFERENCE EXAMPLE 1

[0180] To an aqueous solution of gene recombinant hGH (final hGHconcentration=2 mg/ml) was added ammonium acetate (20-fold molequivalent). The mixture (100 ml) was dropwise added to the inner wallsurface of a distillation flask cooled in a dry ice-ethanol bath using aperistaltic pump over 30 minutes to rapid-freeze the mixture and thefrozen mixture was dried in vacuo to obtain hGH powder. A lacticacid-glycolic acid copolymer (lactic acid/glycolic acid=65/35,viscosity=0.160 dl/g, 1.690 g) and zinc oxide (10 mg) were dissolved indichloromethane (2.7 ml). To the organic solvent solution was added theabove-mentioned hGH powder (359 mg) and the mixture was finelygranulated with Polytron (manufactured by Kinematica). This S/Odispersion was added to a 0.1% aqueous solution of polyvinyl alcohol(800 ml) and the mixture was stirred and emulsified using a homomixer.The mixture was stirred at room temperature for 3 hours to volatilizedichloromethane and centrifuged (about 1,500 rpm) to obtainedmicrocapsules. The microcapsules were then washed twice with distilledwater (400 ml) and lyophilized from D-mannitol (0.2 g) to obtainlyophilized hGH-containing microcapsule powder. Under the sameconditions, six batches of the microcapsules were produced. The yield ofthe lyophilized microcapsule powder obtained was 6.8 g.

REFERENCE EXAMPLE 2

[0181] To an aqueous solution of gene recombinant hGH (final hGHconcentration=2 mg/ml) was added ammonium acetate (20-fold molequivalent). A lactic acid-glycolic acid copolymer (lactic acid/glycolicacid=50/50, viscosity=0.154 dl/g, 1.850 g) and zinc oxide (10 mg) weredissolved in dichloromethane (2.7 ml). To the organic solvent solutionwas added the above-mentioned hGH powder (155 mg) and the mixture wasfinely granulated with Polytron (manufactured by Kinematica). This S/Odispersion was added to a 0.1% aqueous solution of polyvinyl alcohol(800 ml) and the mixture was stirred and emulsified using a homomixer.The mixture was stirred at room temperature for 3 hours to volatilizedichloromethane and centrifuged (about 1,500 rpm) to obtainmicrocapsules. The microcapsules were then washed twice with distilledwater (400 ml) and lyophilized from D-mannitol (0.2 g) to obtainlyophilized hGH-containing microcapsule powder. Under the sameconditions, six batches of the microcapsules were produced. The yield ofthe lyophilized microcapsule powder obtained was 7.6 g.

REFERENCE EXAMPLE 3

[0182] To an aqueous solution of gene recombinant hGH (final hGHconcentration=2 mg/ml) was added ammonium acetate (20-fold molequivalent). The mixture (100 ml) was dropwise added to the inner wallsurface of a distillation flask cooled in a dry ice-ethanol bath using aperistaltic pump over 30 minutes so as to rapid-freeze the mixture andthe frozen mixture was dried in vacuo to obtain hGH powder. A lacticacid-glycolic acid copolymer (lactic acid/glycolic acid=65/35,viscosity=0.160 dl/g, 1.521 g) and zinc oxide (9 mg) were dissolved indichloromethane (2.4 ml). To the organic solvent solution was added theabove-mentioned hGH powder (270 mg) and the mixture was finelygranulated with Polytron (manufactured by Kinematica). This S/Odispersion was added to a 0.1% aqueous solution of polyvinyl alcohol(800 ml) that had been cooled to 18° C., and the mixture was stirred andemulsified using a homomixer. The mixture was stirred at roomtemperature for 3 hours to volatilize dichloromethane and centrifuged(about 1,500 rpm) to obtain microcapsules. To the microcapsulesuspension, which had been obtained by removing the supernatant as muchas possible by aspiration operation, was added a 50% aqueous solution ofethanol (500 ml), and the mixture was stirred gently using a propellerat room temperature for 15 minutes. The mixture was centrifuged (about1,500 rpm) to obtain microcapsules. The microcapsules were then washedtwice with distilled water (400 ml) and lyophilized from D-mannitol (180mg) to give lyophilized hGH-containing microcapsule powder. In order toremove the residual solvent, the powder was dried in vacuo at 46° C. for72 hours to obtain microcapsules.

REFERENCE EXAMPLE 4

[0183] Evaluation of pharmacological effect for human growthhormone-containing microcapsules

[0184] To female SD rats, which had been removed glandula pituitaria atfour-week old, was administered an immunosuppressive agent, tacrolimus(Prograf injection, manufactured by Fujisawa Pharmaceutical Co., Ltd.)to suppress the production of antibodies to hGH. Microcapsules wereadministered to the animal at six-week old, and the body weight, bodylength and concentration of rat insulin-like growth factor I (rIGF-I) inblood serum were measured for 5 weeks. Specifically, the Prografinjection (5 mg) was diluted with saline, and the dilution was injectedsubcutaneously, at the dose of 50 μg/0.2 ml/rat at three days before thefirst administration of microcapsules, immediately after the firstadministration of microcapsules and on the 4th, 7th and 11th days afterthe first administration, and at the dose of 75 μg/0.2 ml/rat on the14th, 18th, 21st, 25th, 28th and 32nd days after the firstadministration, respectively. Furthermore, in order to more physicallynormalize the glandula pituitaria-removed rat, hormone supplementationwas also carried out. A mixed solution of sodium L-thyroxin pentahydrateand hydrocortisone succinate (both are manufactured by Wako purechemical Industries, Ltd) (the final concentrations were 1 μg and 50 μgper 0.2 ml/rat, respectively) was subcutaneously administered threetimes a week, namely, three days before the first administration of themicrocapsules, immediately after the first administration, and on the2nd, 4th, 7th, 9th, 11th, 14th, 16th, 18th, 21st, 23rd, 25th, 28th, 30thand 32nd days after the first administration. The microcapsules weredispersed in a dispersion medium (5% mannitol, 0.5%carboxymethylcellulose sodium, 0.1% Tween 80) so as to be 24 mg hGH/ml,and 0.5 ml of the dispersion was administered subcutaneously to the backof the rat under ether anesthesia. The dose was 12 mg as hGH. After theadministration of microcapsules, the body weight and body length of therat was measured with time up to 35 days. In addition, blood wascollected from the caudal vein with time and blood serum wasfractionated. The concentration of rIGF-I in blood serum was measured byradioimmunoassay (DSL-2900, Diagnostic Systems Laboratories, Inc.).

REFERENCE EXAMPLE 5

[0185] Candesartan (2.0 g), zinc oxide (manufactured by Hakusui ChemicalIndustries, Ltd., 0.37 g) and a lactic acid-glycolic acid copolymer(lactic acid/glycolic acid 75/25 (mol %), weight-average molecularweight 8,700, manufactured by Wako Pure Chemical Industries, Ltd, 3.6 g)were added to a mixed solution of dichloromethane (12.75 ml), methanol(2.25 ml) and acetic acid (0.136 ml), and the mixture was stirred withshaking at room temperature overnight to obtain a homogenous solution.The solution was added to a 0.1% aqueous solution of polyvinyl alcohol(800 ml) containing 20 mM of zinc acetate, which had been previouslyadjusted to 18° C., and an O/W emulsion was prepared using a turbinetype homomixer at 7,000rpm. This O/W emulsion was stirred at roomtemperature for 3 hours to volatilize dichloromethane, methanol andacetic acid, and the oil phase was solidified and collected usingcentrifuge at 3,000 rpm. This was dispersed in distilled water again andfurther centrifuged to wash out free drug and the like. The collectedmicrocapsules were dispersed again by adding distilled water containingmannitol (0.8 g) and lyophilized to obtain a powder. The encapsulationratio of candesartan in microcapsules was 90.9%, and the content ofcandesartan in microcapsules/mannitol powder was 26.5%.

REFERENCE EXAMPLE 6

[0186] One batch was conducted in the following amount for treatment.Candesartan (2.0 g), zinc oxide (manufactured by Hakusui ChemicalIndustries, Ltd., 0.37 g) and a lactic acid-glycolic acid copolymer(lactic acid/glycolic acid 75/25 (mol %), weight-average molecularweight 8,700, manufactured by Wako Pure Chemical Industries, Ltd, 3.6 g)were added to a mixed solution of dichloromethane (12.75 ml), methanol(2.25 ml) and acetic acid (0.136 ml), and the mixture was stirred withshaking at room temperature overnight to obtain a homogenous solution.The solution was added to a 0.1% aqueous solution of polyvinyl alcohol(800 ml) containing 10 mM of zinc acetate, which had been previouslyadjusted to 18° C., and an O/W emulsion was prepared using a turbinetype homomixer at 7,000 rpm. This O/W emulsion was stirred at roomtemperature for 3 hours to volatilize dichloromethane, methanol andacetic acid, and the oil phase was solidified and collected usingcentrifuge at 3,000 rpm. This was dispersed in distilled water again andfurther centrifuged to wash out free drug and the like. Theabove-mentioned operations were conducted by two batches, and themicrocapsules of the two batches were mixed, and the microcapsules weredispersed again by adding distilled water containing mannitol (1.6 g)and lyophilized to obtain a powder. The encapsulation ratio ofcandesartan in microcapsules was 90.7%, and the content of candesartanin microcapsules/mannitol powder was 26.4%.

EXAMPLE 1

[0187] The solvent was removed under the following four conditions using0.3 g of the hGH-containing lyophilized microcapsule powder obtained inReference Example 1, respectively. The microcapsule powder wastransferred into an extraction vessel (volume 10 ml) of a supercriticalfluid extraction apparatus (manufactured by JASCO Corporation). Theapparatus was sealed and heated to a predetermined temperature in athermostat. Carbon dioxide was delivered to a heat exchanger via adelivery pump (SCF-Get) at the bomb pressure (about 6 to 7 MPa) andheated to the given temperature. The pressure applied to the wholesystem was controlled by an automatic pressure regulating valve(SCF-Bpg), and the carbon dioxide was converted into a high-pressuregaseous state at given pressure. The high-pressurized carbon dioxide gaswas then blown into an extraction vessel, and the solvent was removedunder the following four conditions.

[0188] (1) Pressure 2 MPa, temperature 15° C., extraction period 15minutes.

[0189] (2) Pressure 2 MPa, temperature 15° C., extraction period 30minutes.

[0190] (3) Pressure 2 MPa, temperature 15° C., extraction period 45minutes.

[0191] (4) Pressure 2 MPa, temperature 15° C., extraction period 60minutes.

EXAMPLE 2

[0192] The solvent was removed under the following four conditions using0.3 g of the hGH-containing lyophilized microcapsule powder obtained inReference Example 2, respectively. The microcapsule powder wastransferred into an extraction vessel (volume 10 ml) of a supercriticalfluid extraction apparatus (manufactured by JASCO Corporation). Theapparatus was sealed and heated to a given temperature in a thermostat.Carbon dioxide was delivered to a heat exchanger via a delivery pump(SCF-Get) at the bomb pressure (about 6 to 7 MPa) and heated to thepredetermined temperature. The pressure applied to the whole system wascontrolled by an automatic pressure regulating valve (SCF-Bpg), and thecarbon dioxide was converted into a high-pressure gaseous state atpredetermined pressure. The high-pressurized carbon dioxide gas was thenblown into an extraction vessel, and the solvent was removed under thefollowing four conditions.

[0193] (1) Pressure 2 MPa, temperature 15° C., extraction period 30minutes.

[0194] (2) Pressure 2 MPa, temperature 15° C., extraction period 60minutes.

[0195] (3) Pressure 2 MPa, temperature 15° C., extraction period 180minutes.

[0196] (4) Pressure 1 MPa, temperature 150C, extraction period 720minutes.

EXAMPLE 3

[0197] The solvent was removed under the following 18 conditions using0.3 g of the candesartan-containing lyophilized microcapsule powderobtained in Reference Example 5, respectively. The microcapsule powderwas transferred into an extraction vessel (volume 10 ml) of asupercritical fluid extraction apparatus (manufactured by JASCOCorporation). The apparatus was sealed and heated to a predeterminedtemperature using a thermostat. Carbon dioxide was delivered to a heatexchanger via a delivery pump (SCF-Get) at the bomb pressure (about 6 to7 MPa) and heated to the given temperature. The pressure applied to thewhole system was controlled by an automatic pressure regulating valve(SCF-Bpg), and the carbon dioxide was converted into a high-pressuregaseous at the given pressure. The high-pressurized carbon dioxide gaswas then blown into an extraction vessel, and the solvent was removedunder the following 18 conditions.

[0198] (1) Pressure 2.0 MPa, temperature 15° C., extraction period 30minutes.

[0199] (2) Pressure 2.0 MPa, temperature 15° C., extraction period 60minutes.

[0200] (3) Pressure 2.0 MPa, temperature 15° C., extraction period 120minutes.

[0201] (4) Pressure 2.0 MPa, temperature 15° C., extraction period 180minutes.

[0202] (5) Pressure 2.5 MPa, temperature 15° C., extraction period 30minutes.

[0203] (6) Pressure 2.5 MPa, temperature 15° C., extraction period 60minutes.

[0204] (7) Pressure 2.5 MPa, temperature 15° C., extraction period 120minutes.

[0205] (8) Pressure 2.5 MPa, temperature 15° C., extraction period 180minutes.

[0206] (9) Pressure 3.0 MPa, temperature 15° C., extraction period 15minutes.

[0207] (10) Pressure 3.0 MPa, temperature 15° C., extraction period 30minutes.

[0208] (11) Pressure 3.0 MPa, temperature 15° C., extraction period 60minutes.

[0209] (12) Pressure 3.0 MPa, temperature 15° C., extraction period 120minutes.

[0210] (13) Pressure 3.0 MPa, temperature 15° C., extraction period 180minutes.

[0211] (14) Pressure 3.5 MPa, temperature 15° C., extraction period 30minutes.

[0212] (15) Pressure 3.5 MPa, temperature 15° C., extraction period 60minutes.

[0213] (16) Pressure 3.5 MPa, temperature 15° C., extraction period 120minutes.

[0214] (17) Pressure 3.5 MPa, temperature 15° C., extraction period 180minutes.

[0215] (18) Pressure 4.0 MPa, temperature 15° C., extraction period 30minutes.

EXAMPLE 4

[0216] The solvent was removed under the following three conditionsusing the candesartan-containing lyophilized microcapsule powderobtained in Reference Example 6, respectively. The microcapsule powderwas transferred into an extraction vessel (volume 10 ml) of asupercritical fluid extraction apparatus (manufactured by JASCOCorporation). The apparatus was sealed and heated to a predeterminedtemperature using a thermostat. Carbon dioxide was delivered to a heatexchanger via a delivery pump (SCF-Get) at the bomb pressure (about 6 to7 MPa) and heated to a given temperature. The pressure applied to thewhole system was controlled by an automatic pressure regulating valve(SCF-Bpg), and the carbon dioxide was converted into a high-pressuregaseous state at a given pressure. The high-pressurized carbon dioxidegas was then blown into an extraction vessel, and the solvent wasremoved under the following three conditions.

[0217] (1) pressure 3.0 MPa, temperature 15° C., extraction period 60minutes, charged amount of the microcapsules 0.3 g.

[0218] (2) pressure 3.0 MPa, temperature 15° C., extraction period 60minutes, charged amount of the microcapsules 2 g.

[0219] (3) pressure 3.0 MPa, temperature 15° C., extraction period 60minutes, charged amount of the microcapsules 5 g.

TEST EXAMPLE 1

[0220] For the hGH-containing microcapsules and untreated lyophilizedmicrocapsules obtained in Example 1 (1) to (4), the amount of residualdichloromethane (DCM) and hGH content in the microcapsules were measuredby the following method.

[0221] (1) Amount of Residual Dichloromethane (DCM)

[0222] Microcapsules (about 100 mg) were weighed precisely, dissolved indimethylsulfoxide and made up to exactly 5 ml to prepare a samplesolution. Separately, dichloromethane (about 1 g) was weighed preciselyand made up to exactly 20 ml with addition of dimethylsulfoxide. Thissolution was diluted by exactly 10000 times with dimethylsulfoxide toobtain a standardized solution. The sample solution and standardizedsolution (each 1 μl) were tested by gas chromatography under thefollowing conditions, and the peak area of dichloromethane for eachsolution was measured by automatic integration to calculate the amountof dichloromethane.

[0223] Detector: hydrogen flame ionization detector

[0224] Column: OVI-G43 film thickness 3 μm, 0.53 mm i.d.×30 m (Supelco)

[0225] Inlet temperature: 140° C.

[0226] Detector temperature: 260° C.

[0227] Column temperature: 40° C. (10 min retention)→240° C. (35°C./min)→240° C. (20 min retention)→cooling→40° C.

[0228] Carrier gas: helium

[0229] Flow: 35 cm/sec

[0230] (2) hGH Content

[0231] Microcapsule (10 mg) was weighed precisely in a 5 ml messflask,acetonitrile (1.75 ml) was added thereto and the mixture wasultra-sonicated. To the obtained acetonitrile solution was added 150 mMphosphate saline buffer (pH 8.0, 3 ml), and the solution wasultra-sonicated and made up to a given volume with 150 mM phosphatesaline buffer (pH 8.0). An 1 ml portion of the solution was centrifugedat 15000 rpm for 10 min, and the supernatant was filtered using amembrane filter having the pore size of 0.5 μm. This hGH extractsolution was then subjected to size exclusion high-performance liquidchromatography under the following conditions to measure the content ofhGH.

[0232] Column: TSK-gel G2000SWXL, 7.8 mm i.d.×300 mm (manufactured byTosoh Corporation)

[0233] Mobile phase: 0.05 mol/l ammonium hydrogencarbonate solution

[0234] Flow rate: 0.6 ml/min

[0235] The results are shown in Table 1. TABLE 1 Contents of residualdichloromethane and hGH in microcapsules Conditions for treatmentQuality Tempera Residual Content Pressure ture Time DCM of hGH (Mpa) (°C.) (min) (ppm) (%) Untreated microcapsules 738 10.61 (1) 2 15 15 13710.50 (2) 2 15 30  50 10.46 (3) 2 15 45 <32 10.41 (4) 2 15 60 <32 10.52

[0236] As seen from the results in Table 1, the amount of residualdichloromethane in the microcapsules treated with carbon dioxide in ahigh-pressure gaseous state markedly decreased in comparison with theuntreated microcapsules. Furthermore, it was found that the content ofhGH in the microcapsules did not decrease by the treatment with carbondioxide in a high-pressure gaseous state.

TEST EXAMPLE 2

[0237] For the hGH-containing microcapsules and untreated lyophilizedmicrocapsules obtained in Example 1 (1) to (4), the amount of hGHaggregate and hGH related protein in the microcapsules were measured bythe following method.

[0238] (1) hGH Aggregate

[0239] Microcapsules (10 mg) were weighted precisely, acetonitrile (2.5ml) was added thereto, and the sample was dispersed by ultrasonicirradiation. The sample was subsequently irradiated with ultrasonic forabout 2 min, and centrifuged at 3000 rpm for 10 minutes. The supernatantwas removed, and acetonitrile (2.5 ml) was added to the residue. Theresidue was dispersed by ultrasonic irradiation. The dispersion wassubsequently irradiated with ultrasonic for about 2 minutes, andcentrifuged at 3000 rpm for 10 minutes. The supernatant was removed, andthe residue was dried in a desiccator under reduced pressure. To theresidue was added a diluent (phosphate buffer (pH 8.0)/acetonitrilemixed solution (13:7), 1.25 ml), and the sample was dispersed byultrasonic irradiation. The dispersion was subsequently irradiated withultrasonic for about 2 min and filtered with a membrane filter having apore size of 0.5 μm, and the filtrate was used as a sample solution.Separately, hGH reference standard (0.2 ml) was added to a diluent (0.2ml). This solution (0.2 ml) was added to a diluent (4.8 ml) to obtain astandardized solution. The sample solution and standardized solution(each 50 μl) were measured by liquid chromatography under the followingconditions, respectively. The peak area of the peak that elutes earlierthan the retention time of hGH in the sample solution and the peak areaof hGH in the standardized solution were measured by automaticintegration to calculate the content of aggregate. At the same time, adiluent (50 μl) was injected and the peak detected in blank wassubtracted from the calculation.

[0240] Detector: ultraviolet spectrometer (wavelength for measurement:214 nm)

[0241] Column: TSK-gel G2000SWXL, 7.8 mm i.d.×300 mm (manufactured byTosoh Corporation)

[0242] Column temperature: constant temperature about 25° C.

[0243] Mobile phase: 0.05 mol/l ammonium hydrogencarbonate solution

[0244] Flow rate: 0.6 ml/min

[0245] (2) hGH Related Protein

[0246] Microcapsules (40 mg) were precisely weighed and acetonitrile (2ml) was added thereto. The sample was dispersed by ultrasonicirradiation. The dispersion was subsequently irradiated with ultrasonicfor about 2 minutes. Phosphate buffer (pH8.0, 3 ml) was added theretoand the dispersion was irradiated with ultrasonic for about 2 minuteswith occasionally shaking, and centrifuged at 4° C. for 3500 rpm for 10minutes. The supernatant was filtered with a membrane filter having poresize of 0.5 μm, and the filtrate was used as a sample solution.Separately, hGH reference standard (0.1 ml) was added to a diluent(phosphate buffer (pH 8.0)/acetonitrile mixed solution (13:7), 3.9 ml)to obtain a standardized solution. The sample solution and standardizedsolution (each 20 μl) were measured by liquid chromatography under thefollowing conditions, respectively. The peak areas of the substancesother than hGH in the sample solution and the peak area of hGH in thestandardized solution were measured by automatic integration,respectively, to calculate the content of related protein. At the sametime, a diluent (20 μl) was injected and the peak detected in blank wassubtracted from the calculation.

[0247] Detector: ultraviolet spectrometer (measurement wavelength: 220nm)

[0248] Column: PROTEIN C4, 4.6 mm i.d.×250 mm (VYDAC)

[0249] Column temperature: constant temperature about 45° C.

[0250] Mobile phase:(A) 2-amino-2-hydroxymethyl-1,3-propanediol buffer(pH 7.5)/1-propanol mixed solution (19:6)

[0251] (B) 2-amino-2-hydroxymethyl-1,3-propanediol buffer (pH7.5)/1-propanol mixed solution (17:8)

[0252] The solution (A) and solution (B) were flowed in the proportionof (1:1).

[0253] Flow rate: 0.5 ml/min

[0254] The results are shown in Table 2. TABLE 2 Contents of hGHaggregate and related protein in microcapsules Conditions for treatmentQuality Tempera Aggre- Related Pressure ture Time gate protein (Mpa) (°C.) (min) (%) (%) Untreated microcapsules 1.95 5.08 (1) 2 15 15 2.005.42 (2) 2 15 30 1.96 5.47 (3) 2 15 45 1.94 5.50 (4) 2 15 60 1.95 5.26

[0255] As seen from the results in Table 2, the amounts of hGH aggregateand related protein in the microcapsules treated with carbon doixide inthe state of high-pressure gas did not increase in comparison with thoseof the untreated microcapsules.

TEST EXAMPLE 3

[0256] For the hGH-containing microcapsules and untreated lyophilizedmicrocapsules obtained in Example 1 (1) to (4), the mean particlediameter and in vivo initial release percentage of the microcapsuleswere measured by the following method.

[0257] (1) Mean Particle Diameter of Microcapsules

[0258] The mean particle diameter of microcapsules was measured using ameasurement apparatus for particle size distribution (Multisizer II,Coulter Electronics Ltd., Beds, UK).

[0259] (2) In Vivo Initial Release Percentage

[0260] Rats were subjected to immunosuppression treatment withtacrolimus. Prograf injection (manufactured by Fujisawa PharmaceuticalCo., Ltd., 5 mg) was diluted with saline. The dilution wassubcutaneously administered at the dose of, 0.4 mg/0.2 ml/rat (threedays before the first administration of the microcapsules), 0.2 mg/0.2ml/rat (immediately after the first administration of microcapsules, andon the 4th, 7th and 11th days after administration), 0.3 mg/0.2 ml/rat(on the 14th, 18th, 21st, 25th, 28th and 32nd days after the firstadministration), respectively, whereby the production of antibodies tohGH could be suppressed, which allowed the evaluation of theconcentration of hGH in the blood serum of rats for 5 weeks after thefirst administration.

[0261] The microcapsules were dispersed in a dispersion medium (5%mannitol, 0.5% carboxymethylcellulose, 0.1% Tween80) at theconcentration of 16 mg hGH/ml. The obtained dispersion (0.75 ml) wassubcutaneous administered to the back of the rat under ether anesthesia.The dose was 12 mg as hGH. After the administration of themicrocapsules, blood was collected with time from the caudal vein andblood serum was fractionated.

[0262] The measurement of the concentration of hGH in blood serum wasmeasured by immunoradiometric assay (Ab beads HGH, manufactured by EikenChemical Co., Ltd.).

[0263] To the immunosuppressed rats were subcutaneously administered asolution of hGH at the dose of 5, 10 and 20 mg/kg, respectively, andblood was collected with time and the concentration of hGH in bloodserum was measured. AUC was calculated by trapezoid method. From the AUCup to 24 hours after administration of microcapsules, the administeredamount of hGH, the corresponding administered amount of hGH solution inthe case of subcutaneous administration was calculated, which wasdivided by the administered amount of microcapsules (12 mg) to calculatethe initial release percentage.

[0264] The results are shown in Table 3. TABLE 3 Mean particle diameterand initial release percentage of microcapsules Quality Conditions fortreatment Mean Initial Tempera particle release Pressure ture Timediameter percen- (Mpa) (° C.) (min) (μm) tage (%) Untreatedmicrocapsules 36.2 28.3 (1) 2 15 15 34.9 17.0 (2) 2 15 30 35.7 16.7 (3)2 15 45 35.1 13.8 (4) 2 15 60 37.7 24.2

[0265] As seen from the results in Table 3, it was confirmed that themean particle diameter of the microcapsules did not change by thetreatment with carbon dioxide in a high-pressure gaseous state and didnot aggregate. Furthermore, the initial release percentage of themicrocapsules treated with carbon dioxide in a high-pressure gaseousstate markedly decreased in comparison with that of the untreatedmicrocapsules.

TEST EXAMPLE 4

[0266] For the hGH-containing microcapsules and untreated 15 lyophilizedmicrocapsules obtained in Example 2 (1) to (4), the amount of residualdichloromethane (DCM) and hGH content in the microcapsules were measuredby the following method.

[0267] (1) Amount of Residual Dichloromethane (DCM)

[0268] Microcapsules (about 100 mg) were weighed precisely, dissolved indimethylsulfoxide to made up to exactly 5 ml to prepare a samplesolution. Separately, dichloromethane (about 1 g) was measuredprecisely, and dimethylsulfoxide was added thereto to made up to exactly20 ml. This solution was diluted by exactly 10000 times withdimethylsulfoxide to obtain a standardized solution. The sample solutionand standardized solution (each 1 μl) were tested by gas chromatographyunder the following conditions, and the peak area of dichloromethane foreach solution was measured by automatic integration to calculate theamount of dichloromethane.

[0269] Detector: hydrogen flame ionization detector

[0270] Column: OVI-G43 film thickness 3 μm, 0.53 mm i.d.×30 m (Supelco)

[0271] Inlet temperature: 140° C.

[0272] Detector temperature: 260° C.

[0273] Column temperature: 40° C. (10 min retention) 240° C.

[0274] (35° C./min)→240° C. (20 min retention)→cooling 40° C.

[0275] Carrier gas: helium

[0276] Flow: 35 cm/sec

[0277] (2) hGH Content

[0278] Microcapsules (20 mg) were weighed precisely in a 5 ml graduatedflask, and acetonitrile (1.75 ml) was added thereto and the mixture wasultra-sonicated. To the obtained acetonitrile solution was added 150 mMphosphate saline buffer (pH 8.0, 3 ml) and the solution wasultra-sonicated. To the solution was added 150 mM phosphate salinebuffer (pH 8.0) to make up to a given volume. A 1 ml portion of thesolution was centrifuged at 15000 rpm for 10 min, and the supernatantwas filtered using a membrane filter having the pore size of 0.5 μm.This hGH extract solution was then subjected to size exclusionhigh-performance liquid chromatography under the following conditions tomeasure the content of hGH.

[0279] Column: TSK-gel G2000SWXL, 7.8 mm i.d.×300 mm (manufactured byTosoh Corporation)

[0280] Mobile phase: 0.05 mol/l ammonium hydrogencarbonate solution

[0281] Flow rate: 0.6 ml/min

[0282] The results are shown in Table 4. TABLE 4 Contents of residualdichioromethane and hGH in microcapsules Conditions for treatmentQuality Tempera Residual Content Pressure ture Time DCM of hGH (Mpa) (°C.) (min) (ppm) (%) Untreated microcapsules 4283 4.82 (1) 2 15 30 6834.72 (2) 2 15 60 207 4.74 (3) 2 15 180 26 4.68 (4) 1 15 720 1000 4.74

[0283] As is seen from the results in Table 4, the amount of residualdichloromethane in the microcapsules treated with carbon dioxide in ahigh-pressure gaseous state markedly decreased in comparison with thatof the untreated microcapsules. Furthermore, it was found that thecontent of hGH in the microcapsules was not decreased by the treatmentwith carbon dioxide in a high-pressure gaseous state.

TEST EXAMPLE 5

[0284] For the hGH-containing microcapsules and untreated lyophilizedmicrocapsules obtained in Example 2 (1) to (4), the amount of hGHaggregate and hGH related protein in the microcapsules were measured bythe following method.

[0285] (1) hGH Aggregate

[0286] Microcapsules (10 mg) were weighed precisely, acetonitrile (2.5ml) was added thereto, and the sample was dispersed by ultrasonicirradiation. The sample was subsequently irradiated with ultrasonic forabout 2 minutes, and centrifuged at 3000 rpm for 10 minutes. Thesupernatant was removed, and acetonitrile (2.5 ml) was added to theresidue. The residue was dispersed by ultrasonic irradiation. Thedispersion was subsequently irradiated with ultrasonic for about 2 min,and centrifuged at 3000 rpm for 10 minutes. The supernatant was removed,and the residue was dried in a desiccator under reduced pressure. To theresidue was added a diluent (phosphate buffer (pH 8.0)/acetonitrilemixed solution (13:7), 1.25 ml), and the sample was dispersed byultrasonic irradiation. The dispersion was subsequently irradiated withultrasonic for about 2 minutes and filtered with a membrane filterhaving pore size of 0.5 μm, and the filtrate was used as a samplesolution. Separately, hGH reference standard (0.2 ml) was added to adiluent (0.2 ml). This solution (0.2 ml) was added to a diluent (4.8 ml)to obtain a standardized solution. The sample solution and standardizedsolution (each 50 μl) were measured by liquid chromatography under thefollowing conditions, respectively. The peak area of the peak thateluted earlier than the retention time of hGH in the sample solution andthe peak area of hGH in the standardized solution were measured byautomatic integration to calculate the content of aggregate. At the sametime, a diluent (50 μl) was injected and the peak detected in blank wassubtracted from the calculation.

[0287] Detector: ultraviolet spectrometer (wavelength for measurement:214 nm)

[0288] Column: TSK-gel G2000SWXL, 7.8 mm i.d.×300 mm (manufactured byTosoh Corporation)

[0289] Column temperature: constant temperature about 25° C.

[0290] Mobile phase: 0.05 mol/l ammonium hydrogencarbonate solution

[0291] Flow rate: 0.6 ml/min

[0292] (2) hGH Related Protein

[0293] Microcapsules (40 mg) were precisely weighed and acetonitrile (2ml) was added thereto. The sample was dispersed by ultrasonicirradiation. The dispersion was subsequently irradiated with ultrasonicfor about 2 minutes. Phosphate buffer (pH 8.0, 3 ml) was added theretoand the dispersion was irradiated with ultrasonic for about 2 minuteswith occasionally shaking, and centrifuged at 4° C. for 3500 rpm for 10min. The supernatant was filtered with a membrane filter having poresize of 0.5 μm, and the filtrate was used as a sample solution.Separately, hGH reference standard (0.1 ml) was added to a diluent(phosphate buffer (pH 8.0)/acetonitrile mixed solution (13:7), 3.9 ml)to obtain a standardized solution. The sample solution and standardizedsolution (each 20 μl) were measured by liquid chromatography under thefollowing conditions, respectively. The peak areas of the substancesother than hGH in the sample solution and the peak area of hGH in thestandardized solution were measured by automatic integration,respectively, to calculate the content of related protein. At the sametime, a diluent (20 μl) was injected and the peak detected in blank wassubtracted from the calculation.

[0294] Detector: ultraviolet spectrometer (measurement wavelength:untreated microcapsules.

TEST EXAMPLE 6

[0295] For the hGH-containing microcapsules and untreated lyophilizedmicrocapsules obtained in Example 2 (1) to (4), the mean particlediameter and in vivo initial release percentage of the microcapsuleswere measured by the following method.

[0296] (1) Mean Particle Diameter of Microcapsules

[0297] The mean particle diameter of microcapsules was measured using ameasurement apparatus for particle size distribution (Multisizer II,Coulter Electronics Ltd., Beds, UK).

[0298] (2) In Vivo Initial Release Percentage

[0299] Rats were subjected to immunosuppression treatment withtacrolimus. Prograf injection (manufactured by Fujisawa PharmaceuticalCo., Ltd., 5 mg) was diluted with saline. The dilution wassubcutaneously administered in the dose of, 0.4 mg/0.2 ml/rat (threedays before administration of the microcapsules), 0.2 mg/0.2 ml/rat(immediately after the first administration of microcapsules, and on the4th, 7th, 11th, 14th and 18th days after the first administration),respectively. The microcapsules were dispersed in a dispersion medium(5% mannitol, 0.5% carboxymethylcellulose, 0.1% Tween80) at theconcentration of 8 mg hGH/ml. The obtained dispersion (0.75 ml) wassubcutaneous administered to the back of the rat under ether anesthesia.The dose was 6 mg as hGH. After the administration of the microcapsules,blood was sequentially taken from the caudal vein and blood serum wasfractionated.

[0300] The concentration of hGH in blood serum was measured byimmunoradiometric assay (Ab beads HGH, Eiken Chemical Co., Ltd.).

[0301] To the immunosuppressed rat was subcutaneously administered asolution of hGH at the dose of 5, 10 and 20 mg/kg, respectively, andblood was collected with time and the concentration of hGH in bloodserum was measured. AUC was calculated by trapezoid method. From the AUCup to 24 hr after administration of microcapsules, the administeredamount of hGH, the corresponding administered amount of hGH solution inthe case of subcutaneous administration was calculated, which wasdivided by the administered amount of microcapsules (6 mg) to calculatethe initial release percentage.

[0302] The results are shown in Table 6.

[0303] Microcapsules (about 100 mg) were weighed precisely, dissolved indimethylsulfoxide to make up to exactly 5 ml to prepare a samplesolution. Separately, dichloromethane (about 1 g) was weighed precisely,and dimethylsulfoxide was added thereto to make up to exactly 20 ml.This solution was diluted by exactly 10000 times with dimethylsulfoxideto obtain a standardized solution. The sample solution and standardizedsolution (each 1 μl) were tested by gas chromatography under thefollowing conditions, and the peak area of dichloromethane for eachsolution was measured by automatic integration to calculate the amountof dichloromethane.

[0304] Detector: hydrogen flame ionization detector

[0305] Column: OVI-G43 film thickness 3 μm, 0.53 mm i.d.×30 m (Supelco)

[0306] Inlet temperature: 140° C.

[0307] Detector temperature: 260° C.

[0308] Column temperature: 40° C. (10 min retention)→260° C. (35°C./min) (10 min retention)

[0309] Carrier gas: helium

[0310] Flow: 35 cm/sec

[0311] (2) Candesartan Content

[0312] Microcapsules (5 to 10 mg) were weighed precisely in a centrifugetube, HPLC mobile phase (30 ml) was added thereto and the mixture wasstirred with shaking for 1 hour.

[0313] The mixture was then centrifuged at 2950 rpm for 10 minutes, andthe supernatant was filtered with a membrane filter having the pore sizeof 0.5 μm. This candesartan extract solution was then subjected toreverse phase high-performance liquid chromatography under the followingconditions to measure the content of candesartan.

[0314] Column : Inertsil ODS-3 (4.6 mm×150 mm, manufactured by GLscience)

[0315] Mobile phase: 0.1M KH₂PO₄/AcCN/MeOH/AcOH=50/35/15/1 (v/v)

[0316] Flow rate:1 ml/min

[0317] Detection: UV wavelength 254 nm

[0318] The results are shown in Table 7. TABLE 7 Contents of residualdichloromethane and candesartan in microcapsules Quality Conditions fortreatment Residual Tempera- Charged dichloro- Drug Pressure ture Timeamount methane content (Mpa) (° C.) (min) (g) (ppm) (%) Untreatedmicrocapsules 18026 26.5 (1) 2.0 15 30 0.3 826 26.0 (2) 2.0 15 60 0.3230 26.0 (3) 2.0 15 120 0.3 106 26.5 (4) 2.0 15 180 0.3 0 26.3 (5) 2.515 30 0.3 411 26.1 (6) 2.5 15 60 0.3 375 26.9 (7) 2.5 15 120 0.3 0 26.9(8) 2.5 15 180 0.3 0 26.5 (9) 3.0 15 15 0.3 6923 26.8 (10) 3.0 15 30 0.32993 26.2 (11) 3.0 15 60 0.3 0 26.8 (12) 3.0 15 120 0.3 0 26.9 (13) 3.015 180 0.3 0 26.8 (14) 3.5 15 30 0.3 3926 26.3 (15) 3.5 15 60 0.3 0 27.1(16) 3.5 15 120 0.3 321 26.9 (17) 3.5 15 180 0.3 0 26.5 (18) 4.0 15 300.3 1148 27.1

[0319] As is seen from the results in Table 7, the amount of residualdichloromethane in the microcapsules treated with carbon dioxide in thehigh-pressure gaseous state markedly decreased in comparison with thatof the untreated microcapsules. Furthermore, it was confirmed that thecontent of candesartan in the microcapsules was not decreased by thetreatment with carbon dioxide in the state of high-pressure gas.

TEST EXAMPLE 8

[0320] For the candesartan-containing microcapsules and untreatedlyophilized microcapsules obtained in Example 4 (1) to (3), the amountof residual dichloromethane (DCM) and candesartan content in themicrocapsules were measured by the similar method to that of TestExample 7.

[0321] The results are shown in Table 8. TABLE 8 Contents of residualdichloromethane and candesartan in microcapsules Quality Conditions fortreatment Residual Tempera- Charged dichloro- Drug Pressure ture Timeamount methane content (Mpa) (° C.) (min) (g) (ppm) (%) Untreatedmicrocapsules 24692 26.4 (1) 3.0 15 60 0.3 116 26.8 (2) 3.0 15 60 2 026.4 (3) 3.0 15 60 5 137 27.7

[0322] As is seen from the results in Table 8, the amount of residualdichloromethane in the microcapsules treated with carbon dioxide in thehigh-pressure gaseous state markedly decreased in comparison with thatof the untreated microcapsules. Furthermore, it was confirmed that thecontent of candesartan in the microcapsules did not decrease by thetreatment with carbon dioxide in a high-pressure gaseous state.

INDUSTRIAL APPLICABILITY

[0323] According to the present invention, in a method for producing asustained-release preparation, by forming a solid material containing abioactive substance and a polymer and contacting the solid material withhigh-pressure gas, it is possible to produce a sustained-releasepreparation which is a medicament having such very superior clinicalproperties that the excess amount of initial release of the bioactivesubstance immediately after administration is markedly suppressed, aconstant amount of the bioactive substance is being released fromimmediately after administration over a long period of time, and thedenaturation of the bioactive substance and the residual organic solventare extremely decreased. Furthermore, by modifying the method forremoving a solvent, the treatment period required for the removal of thesolvent has been markedly decreased.

1. A method for producing a preparation containing a bioactive substance, which comprises forming a solid material containing the bioactive substance and a polymer, and contacting the solid material with high-pressure gas.
 2. The method according to claim 1, wherein the bioactive substance is that being unstable to heat or solvents.
 3. The method according to claim 1, wherein the bioactive substance is a bioactive peptide having a molecular weight of about 2,000 to about 500,000.
 4. The method according to claim 1, wherein the bioactive substance is a bioactive peptide having a molecular weight of about 5,000 to about 500,000.
 5. The method according to claim 4, wherein the bioactive substance is human growth hormone.
 6. The method according to claim 1, wherein the bioactive substance is a non-peptidic compound.
 7. The method according to claim 6, wherein the non-peptidic compound is a compound having an oxygen atom in the molecule.
 8. The method according to claim 6, wherein the non-peptidic compound is a compound having an ether bond or a carbonyl group.
 9. The method according to claim 6, wherein the non-peptidic compound is a compound represented by the formula (I):

wherein R¹ represents a group capable of forming an anion or a group which may be converted into said group, X represents that the phenylene group and the phenyl group are linked directly or via a spacer of an atomic chain having two or less atom(s), n represents an integer of 1 or 2, ring A represents a benzene ring which may be further substituted, R² represents a group capable of forming an anion or a group which may be converted into said group, R³ represents a hydrocarbon residue which may link via a heteroatom and may be substituted, or a salt thereof.
 10. The method according to claim 6, wherein the non-peptidic compound is losartan, eprosartan, candesartan cilexetil, candesartan, valsartan, telmisartan, irbesartan, tasosartan or olmesartan.
 11. The method according to claim 6, wherein the non-peptidic compound is candesartan.
 12. The method according to claim 1, wherein the polymer is biodegradable.
 13. The method according to claim 12, wherein the biodegradable polymer is a homopolymer or a copolymer of α-hydroxycarboxylic acids, or a mixture thereof.
 14. The method according to claim 13, wherein the biodegradable polymer is a homopolymer or a copolymer of lactic acid/glycolic acid having a composition ratio of lactic acid/glycolic acid of about 100/0 to about 40/60 mol %.
 15. The method according to claim 13, wherein the biodegradable polymer is a homopolymer of lactic acid.
 16. The method according to claim 12, wherein the weight-average molecular weight of the biodegradable polymer is about 3,000 to about 50,000.
 17. The method according to claim 1, wherein the solid material is contacted with high-pressure gas at a temperature range of about +20° C. to about −60° C. based on the glass transition temperature of the polymer.
 18. The method according to claim 17, wherein the solid material is contacted with high-pressure gas at a temperature range of about +0° C. to about −40° C. based on the glass transition temperature of the polymer.
 19. The method according to claim 1, wherein the period for contacting the solid material with high-pressure gas is about 5 minutes to about 48 hours.
 20. The method according to claim 19, wherein the period for contacting the solid material with high-pressure gas is about 10 minutes to about 12 hours.
 21. The method according to claim 1, wherein the high-pressure gas is inert to the bioactive substance and polymer.
 22. The method according to claim 21, wherein the high-pressure gas is carbon dioxide.
 23. The method according to claim 1, wherein the pressure of the high-pressure gas is about 1 MPa to about 7 MPa.
 24. The method according to claim 23, wherein the pressure of the high-pressure gas is about 1 MPa to about 4 MPa.
 25. The method according to claim 1, wherein the preparation is sustained-release microcapsules.
 26. The method according to claim 25, wherein the sustained-release microcapsules are obtained by in-water drying method.
 27. A preparation obtained by the method according to claim
 1. 28. Sustained-release microcapsules obtained by the method according to claim
 25. 29. An injectable preparation containing the sustained-release microcapsules according to claim
 28. 30. A method for suppressing the initial release of a bioactive substance, which comprises forming a solid material containing said bioactive substance and a polymer, and contacting the solid material with high-pressure gas.
 31. A method for suppressing the denaturation of a bioactive substance, which comprises forming a solid material containing said bioactive substance and a polymer, and contacting the solid material with high-pressure gas. 